Oncoprotein protein kinase

ABSTRACT

The present invention provides an isolated polynucleotide encoding a c-Jun peptide consisting of about amino acid residues 33 to 79 as set fort in SEQ ID NO: 10 or conservative variations thereof. The invention also provides a method for producing a peptide of SEQ ID NO:1 comprising (a) culturing a host cell containing a polynucleotide encoding a c-Jun peptide consisting of about amino acid residues 33 to 79 as set forth in SEQ ID NO: 10 under conditions which allow expression of the polynucleotide; and (b) obtaining the peptide of SEQ ID NO:1.

This application is a continuation prior U.S. application Ser. No.09/150,201; filed on Sep. 8, 1998, issuing on Dec. 14, 1999 as U.S. Pat.No. 6,001,584, which is a divisional (and claims the benefit of priorityunder 35 USC §120) of U.S. application Ser. No. 08/799,913, filed Feb.13, 1997, now U.S. Pat. No. 5,804,399, which is a continuation of08/444,393, filed May 19, 1995, now U.S. Pat. No. 5,605,808, which is adivisional of U.S. patent application Ser. No. 08/276,860, filed Jul.18, 1994, now U.S. Pat. No. 5,593,884, which is a continuation-in-partapplication of U.S. patent application Ser. No. 08/220,602, filed Mar.25, 1994, which is a continuation-in-part of U.S. patent applicationSer. No. 08/094,533, filed Jul. 19, 1993, now U.S. Pat. No. 5,534,419,the contents of which are incorporated by reference in their entiretyherein.

This invention was made with support by Howard Hughes Medical Instituteand Government support under Grant No. DE86ER60429, awarded by theDepartment of Energy and Grant No. CA-50528 and CA-58396, awarded by theNational Institute of Health. The Government has certain rights in thisinvention. Also supported by the Howard Hughes Medical Institute.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the field of protein kinases,oncogenes and oncoproteins and, specifically, to a protein kinase whichbinds, phosphorylates and potentiates the c-Jun N-terminal activationdomain.

2. Description of Related Art

A number of viral and cellular genes have been identified as potentialcancer genes, collectively referred to as oncogenes. The cellularhomologs of viral oncogenes, the proto-oncogenes or c-oncogenes, act inthe control of cell growth and differentiation or mediate intracellularsignaling systems. The products of oncogenes are classified according totheir cellular location, for example, secreted, surface, cytoplasmic,and nuclear oncoproteins.

Proto-oncogenes which express proteins which are targeted to the cellnucleus make up a small fraction of oncogenes. These nuclearproto-oncoproteins typically act directly as transactivators andregulators of RNA and DNA synthesis. Nuclear oncogene products have theability to induce alterations in gene regulation leading to abnormalcell growth and ultimately neoplasia. Examples of nuclear oncogenesinclude the myc, ski, myb, fos and jun genes.

The c-Jun protein, encoded by the c-jun proto-oncogene, is an importantcomponent of the dimeric, sequence specific, transcriptional activator,AP-1. Like other transcriptional activators, c-Jun contains twofunctional domains, including a DNA binding domain and a transactivationdomain. The DNA binding domain is located at the C-terminus and is aBZip structure which consists of conserved basic (B) and leucine zipper(Zip) domains that are required for DNA binding and dimerization,respectively. The N-terminus contains the transactivation domain.Although c-Jun expression is rapidly induced by many extracellularsignals, its activity is also regulated post-translationally by proteinphosphorylation. Phosphorylation of sites clustered next to c-Jun's DNAbinding domain inhibits DNA binding (Boyle, et al., Cell, 6:573, 1991;Lin, et al., Cell, 70:777, 1992). Phosphorylation of two other sites,Ser 63 and Ser 73, located within the transactivation domain, potentatesc-Jun's ability to activate transcription (Binetruy, et al., Nature35:122, 1991; Smeal, et al., Nature 34:494, 1991). Phosphorylation ratesof these sites are low in non-stimulated cells and are rapidly increasedin response to growth factors such as-platelet derived growth factor(PDGF) or v-Sis, or expression of oncogenically activated Src, Ras andRaf proteins. In myeloid and lymphoid cells, phosphorylation of thesesites is stimulated by the phorbol ester, TPA, but not in fibroblastsand epithelial cells. These differences may be due to different modes ofHa-ras regulation in lymphoid cells versus fibroblasts.

Many proteins cooperate with each other in the activation oftranscription from specific promoters. Through this cooperation, a genecan be transcribed and a protein product generated. Members of the Fosproto-oncogene family, along with members of the Jun gene family, formstable complexes which bind to DNA at an AP-1 site. The AP-1 site islocated in the promoter region of a large number of genes. Binding ofthe Fos/Jun complex activates transcription of a gene associated with anAP-1 site. In cells that have lost their growth regulatory mechanisms,it is believed that this Fos/Jun complex may “sit” on the AP-1 site,causing overexpression of a particular gene. Since many proliferativedisorders result from the overexpression of an otherwise normal gene,such as a proto-oncogene, it would be desirable to identify compositionswhich interfere with the excessive activation of these genes.

For many years, various drugs have been tested for their ability toalter the expression of genes or the translation of their messages intoprotein products. One problem with existing drug therapy is that ittends to act indiscriminately and affects healthy cells as well asneoplastic cells. This is a major problem with many forms ofchemotherapy where there are severe side effects primarily due to theaction of toxic drugs on healthy cells.

In view of the foregoing, there is a need to identify specific targetsin the abnormal cell which are associated with the overexpression ofgenes whose expression products are implicated in cell proliferativedisorders, in order to decrease potential negative effects on healthycells. The present invention provides such target.

SUMMARY OF THE INVENTION

The present invention provides a novel protein kinase (JNK) whichphosphorylates the c-Jun N-terminal activation domain. JNK1 ischaracterized by having a molecular weight of 46 kD (as determined byreducing SDS-polyacrylamide gel electrophoresis (PAGE)) and havingserine and threonine kinase activity. Specifically, JNK1 phosphorylatesserine residues 63 and 73 of c-Jun.

Since the product of the jun proto-oncogene is a transactivator proteinwhich binds at AP-1 sites, regulation of c-Jun activation may beimportant in affecting normal gene expression and growth control in acell. The discovery of JNK provides a means for identifying compositionswhich affect JNK activity, thereby affecting c-Jun activation andsubsequent activation of genes associated with AP-1 sites.

The identification of JNK now allows the detection of the level ofspecific kinase activity associated with activation of c-Jun and AP-1.In addition, the invention provides a method of treating a cellproliferative disorder associated with JNK by administering to a subjectwith the disorder, a therapeutically effective amount of a reagent whichmodulates JNK activity.

The invention also provides a synthetic peptide comprising the JNKbinding region on c-Jun which corresponds to amino acids 33-79. Thepeptide is useful as a competitive inhibitor of the naturally occurringc-Jun in situations where it is desirable to decrease the amount ofc-Jun action by JNK.

The invention also describes JNK2, a novel protein kinase with activitysimilar to JNK1 and having a molecular weight of 55 kD.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an SDS-PAGE of nuclear and cytosolic extracts from FR3T3(−) and Ha-ras-transformed FR3T3 (+) cells after incubation with ³²P-ATPand GST-cJun (wt), GSTcJun(Ala63/73) or GST.

FIG. 2 shows an SDS-PAGE of A) HeLaS3 cells either untreated orirradiated with UV light and B) Jurkat cells either untreated orincubated with TPA. Cell extracts were incubated with ³²P-ATP andGST-cJun (wt), GSTcJun-(Ala63/73) or GST.

FIG. 3 shows phosphopeptide mapping of GST-cJun and c-Jun phosphorylatedby JNK. 3(A) shows maps of GSTcJun and (B) shows maps of c-Jun.

FIG. 4 A shows an SDS-PAGE of phosphorylated proteins after elution ofJNK from GSTc-Jun after washes of NaCl, Urea, Guanidine-HCl(GuHCl) orSDS. FIG. 4B shows an SDS-PAGE of phosphorylated c-Jun after GSTcJun(wt)was covalently linked to GSH-beads and incubated with whole cell extractof TPA-stimulated Jurkat cells.

FIG. 5 shows an in-gel kinase assay. GSTcJun-GSH agarose beads wereincubated with cell extracts from A) TPA-stimulated Jurkat cells on SDSgels that were polymerized in the absence (−) or presence (+) of GSTcJun(wt); B) extracts of unstimulated or UV stimulated HeLa cells andunstimulated or TPA-stimulated Jurkat cells; and C) extracts from cellsof logarithmically growing K562 and Ha-ras-transformed FR3T3,TPA-stimulated Jurkat and U937 cells and UV-irradiated HeLa, F9 and QT6cells.

FIG. 6A is a protein gel of various GST c-Jun fusion proteins;

FIG. 6B shows an SDS-PAGE of whole cell extracts of UV-irradiated HelaS3 cells after passage over GSH-beads containing the GST fusion proteinsas shown in FIG. 6A;

FIG. 6C shows an SDS-PAGE of phosphorylated GSTcJun fusion proteinseluted with 1 MNaCl from GSH-agarose beads.

FIG. 7A shows patterns of GST, GSTcJun and GSTcJun as expressed in E.coli; FIG. 7B shows the phosphorylated proteins of 7A from extracts ofTPA-activated Jurkat cells incubated with GSH-beads;

FIG. 7C shows cJun protein after phosphorylation with protein bound toGSTcJun and GSTcJun beads.

FIG. 8 shows CAT activity in cells containing various portions of thec-Jun activation domain (cJ=AA1-223; 33=AA33-223; 43=AA43-223;56=AA56-223; A63,73=AA1-246(Ala63/73)) and a CAT reporter in the absenceor presence of A) Ha-ras or B) UV treatment.

FIG. 9 shows SDS-PAGE analyses of ³²P and ³⁵S labelled F9 cellstransfected with v-Jun and c-Jun in the absence or presence of A) Ha-rasor B) UV exposure.

FIG. 10 shows the nucleotide and deduced amino acid sequence of c-Jun.The arrows represent amino acid residues 33-79.

FIG. 11A shows a Northern blot of total cytoplasmic RNA from Jurkatcells. Cells were incubated with 50 ng/ml TPA (T), 1 μml A23187 (A) or100 ng/ml cycdosporin A (CsA) for 40 minutes, either alone or incombination, as indicated. Levels of c-jun, jun-B, jun-D, c-fos andα-tubulin expression were determined by hybridization to random primedcDNA probes.

FIG. 11B shows Jurkat cells after incubatation with soluble anti-CD3(OKT3), 2 μg/ml soluble anti-CD28 (9.3) or a combination of 50 ng/ml TPAand 1 μg/ml A23817 (T/A) as indicated for 40 minutes. Total cytoplasmicRNA was isolated and 10 μg samples were analyzed using c-Jun, jun-D andc-fos probes. IL-2 induction by the same treatments was measured after 6hours of stimulation by blot hybridization using IL-2 and α-tubulinspecific probes.

FIG. 11C shows Jurkat cells transfected with 10 μg of either −73Col-LUCor −60Col-LUC reporter plasmids. 24 hours after transfection, the cellswere aliquoted into 24 well plates and incubated for 9 hours with 50ng/ml TPA, 1 μg/ml A23187 or 100 ng/ml CsA, either alone or incombination, as indicated. The cells were harvested and luciferaseactivity was determined. The results shown are averages of threeexperiments done in triplicates.

FIG. 12A shows Jurkat cells (10⁶ cells per lane) transfected with 0.5 ugof a SRα-cJun expression vector and 24 hours later were labeled for 3hours with ³²P-orthophosphate (1 mCi/ml). After 15 minutes, treatmentwith 50 ng/ml TPA (T), 1 μg/ml A23187 (A) and 100 ng/ml CsA, eitheralone or in combination, as indicated, the cells were lysed in RIPAbuffer and c-jun was isolated by immunoprecipitation and analyzed bySDS-PAGE The c-Jun bands are indicated.

FIG. 12B shows 2×10⁷ Jurkat cells labeled for 3 hours with either³⁵S-methionine (900 μCi/ml) or ³²P-orthophosphate (1 mCi/ml). After 15minutes incubation with 50 ng/ml TPA+1 μg/ml A23178 (T/A) in the absenceor presence of and 100 ng/ml CsA or no addition, as indicated, the cellswere lysed in RIPA buffer and c-Jun isolated by immunoprecipitation andanalyzed by SDS-PAGE. The c-Jun band is indicated.

FIG. 12C shows all of the c-Jun specific protein bands shown in FIG. 12Aisolated from equal numbers of cells excised from the gel and subjectedto tryptic phosphopeptide mapping. Shown is a typical result (thisexperiment was repeated at least three times). N-nonstimulated cells:T-cells treated with 50 ng/ml TPA; T/A: cells treated with 50 ng/ml TPAand 1 μg/ml A23187; T/A+CsA: cells treated with T/A and 100 ng/ml CsA.a,b,c,x and y correspond to the various tryptic phosphopeptides ofc-Jun, previously described by Boyle, et al., (Cell, 64:573-584, 1991)and Smeal, et al., (Nature, 354:494-496, 1991). T1 and T2 correspond tothe minor phosphorylation sites; Thr91, 93 and 95 (Hibi, et al., Genes &Dev., 7:000, 1993).

FIG. 13A shows whole cell extracts (WCE) of Jurkat cells incubated withTPA (T, 50 ng/ml), A23187 (A, 1 μg/ml) or CsA (100 ng/ml) for 15minutes, alone or in combination, and separated by SDS-PAGE (100 μgprotein/lane) on gels that were cast in the absence or presence ofGST-cJun (1-223). The gels were subjected to renaturation protocol andincubated in kinase buffer, containing γ-³²P-ATP. The protein bandscorresponding to the 55 kD and 46 kD forms of JNK are indicated.

FIG. 13B shows WCE (50 μg) of Jurkat cells treated as described abovewere incubated with 5 μl of GSH agarose beads coated with 10 μg GST-cJun(1-223) for 12 hours at 4° C. After extensive washing, the beads wereincubated in kinase buffer containing γ-³²P-ATP for 20 minutes at 30°C., after which the proteins were dissociated by incubation in SDSsample buffer and separated by SDS-PAGE The 49 kD band corresponds toGST-cJun (1-223).

FIG. 13C shows WCE (20 μg) of Jurkat cells treated as described in FIG.13A and incubated with GST-cJun(1-223)-GSH agarose beads. The boundfraction was eluted in SDS sample buffer and separated by SDS-PAGE on agel containing GST-cJun(1-223). The gel was renatured and incubated inkinase buffer containing γ-³²P-ATP to label the JNK polypeptides.

FIG. 14 shows a phosphorylation assay of cultures of FR3T3, CV-1, PC12and mouse thymocytes were incubated for 15 minutes in the presence ofTPA (50 ng/ml, T), A23817 (1 μg/ml, A) and/or CsA (100 ng/ml), asindicated. WCE prepared from 2-4×10⁵ cells for the established celllines and 1.5×10⁶ cells for primary thymocytes were incubated withGSTcJun(1-223)-GSH agarose beads. After washing, JNK activity wasdetermined by solid-state phosphorylation assay.

FIG. 15 shows WCE (5 ug) of Jurkat (panel A) or mouse thymocytes (panelC) incubated with 1 μg of kinase-defective ERK1 in kinase buffercontaining γ-³²P-ATP for 20 minutes. The phosphorylated proteins wereseparated by SDS-PAGE and the band corresponding to the mutant ERK1 isindicated. WCE (20 μg) of Jurkat (panel A) or mouse thymocytes (panel C)that were treated as described above were immunoprecipitated withanti-ERK antibodies. The immune complexes were washed and incubated inkinase buffer containing γ-³²P-ATP and 2 μg MBP for 15 minutes at 30° C.The phosphorylated proteins were separated by SDS-PAGE. The bandcorresponding to phosphorylated MBP is indicated in panels B and D.

FIG. 16A shows Jurkat cells (1×10⁷) incubated for 15 minutes with eithernormal mouse serum, 1 μg/ml anti-CD3 and/or 2 μg/ml anti-CD28, in theabsence or presence of 100 ng/ml CsA, as indicated. WCE were preparedand 100 μg samples were analyzed for JNK activation using an in-gelkinase assay.

FIG. 16B shows WCE (50 μg) of Jurkat cells treated as described for FIG.16A incubated with GSTcJun(1-223)-GSH agarose beads and assayed for JNKactivity using the solid-state kinase assay. The same WCE (20 μg) wereimmunoprecipitated with anti-ERK2 antibodies and assayed for MBP-kinaseactivity.

FIG. 16C shows WCE (50 μg) of Jurkat cells treated as described in FIG.16A with various stimuli alone or their combinations were incubated withGSTcJun(1-223)-GSH agarose beads and assayed for JNK activity usingsolid-state kinase assay. The same samples (20 μg) were also assayed forMBP-kinase activity as described in FIG. 16B.

FIG. 17A shows Jurkat cells (2×10⁶ cells per point) labeled with 0.4 mCiof ³²P-orthophosphate for 3 hours and incubated with nonspecificantibody (1 μg/ml mouse IgG; control), 1 μg/ml anti-CD3, 2 μg/mlanti-CD28, 10 ng/ml TPA or 500 ng/ml A23187 (A), as indicated. After 2minutes, the cells were harvested, lysed and Ha-Ras was isolated byimmunoprecipitation. The guanine nucleotide bound to Ha-Ras wasextracted, separated by thin layer chromatography and quantitiated. Thevalues shown represent the averages of two separate experiments done induplicates.

FIG. 17B shows Jurkat cells labeled with ³²P-orthophosphate andstimulated with either TPA or ant-CD3. At the indicated time points, thecells were harvested and the GTP content of Ha-Ras was determined.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel protein kinase (JNK) which bindsto a well-defined region of the c-Jun proto-oncoprotein andphosphorylates two sites within its activation domain. Thephosphorylation of these sites increases the ability of c-Jun tostimulate transcription and mediate oncogenic transformation.

The activity of c-Jun is regulated by phosphorylation. Various stimuli,including transforming oncogenes and UV light, induce thephosphorylation of serines 63 and 73 in c-Jun's N-terminal activationdomain, thereby potentiating its transactivation function. The inventionrelates to an isolated polypeptide characterized by having a molecularweight of 46 kD as determined by reducing SDS-PAGE, having serine andthreonine kinase activity and capable of phosphorylating the c-JunN-terminal activation domain. This protein is referred to JNK1. Inaddition, a second JNK protein (55 kD) referred to as JNK2, isdescribed.

The term “isolated” means any JNK polypeptide of the present invention,or any gene encoding a JNK polypeptide, which is essentially free ofother poly-peptides or genes, respectively, or of other contaminantswith which the JNK polypeptide or gene might normally be found innature.

The invention includes a functional polypeptide, JNK and functionalfragments thereof. As used herein, the term “functional polypeptide”refers to a polypeptide which possesses a biological function oractivity which is identified through a defined functional assay andwhich is associated with a particular biologic, morphologic, orphenotypic alteration in the cell. The biological function, for example,can vary from a polypeptide fragment as small as an epitope to which anantibody molecule can bind to a large polypeptide which is capable ofparticipating in the characteristic induction or programming ofphenotypic changes within a cell. An enzymatically functionalpolypeptide or fragment of JNK possesses c-Jun N-terminal activationdomain kinase activity. A “functional polynucleotide” denotes apolynucleotide which encodes a functional polypeptide as describedherein.

Minor modifications of the JNK primary amino acid sequence may result inproteins which have substantially equivalent activity as compared to theJNK polypeptide described herein. Such modifications may be deliberate,as by site-directed mutagenesis, or may be spontaneous. All of thepolypeptides produced by these modifications are included herein as longas the kinase activity of JNK is present. Further, deletion of one ormore amino acids can also result in a modification of the structure ofthe resultant molecule without significantly altering its kinaseactivity. This can lead to the development of a smaller active moleculewhich would have broader utility. For example, it is possible to removeamino or carboxy terminal amino acids which may not be required for JNKkinase activity.

The JNK polypeptide of the invention also includes conservativevariations of the polypeptide sequence. The term “conservativevariation” as used herein denotes the replacement of an amino acidresidue by another, biologically similar residue. Examples ofconservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as thesubstitution of arginine for lysine, glutamic for aspartic acids, orglutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide.

The invention also provides a synthetic peptide which binds to the c-JunN-terminal kinase, JNK. The amino acid sequence of SEQ ID NO: 1, andconservative variations, comprises the synthetic peptide of theinvention. This sequence represents amino acids 33-79 of c-Junpolypeptide (Angel, et al., Nature, 332(6160):166, 1988) As used herein,the term “synthetic peptide” denotes a peptide which does not comprisean entire naturally occurring protein molecule. The peptide is“synthetic” in that it may be produced by human intervention using suchtechniques as chemical synthesis, recombinant genetic techniques, orfragmentation of whole antigen or the like.

Peptides of the invention can be synthesized by such commonly usedmethods as t-BOC or FMOC protection of alpha-amino groups. Both methodsinvolve stepwise syntheses whereby a single amino acid is added at eachstep starting from the C terminus of the peptide (See, Coligan, et al.,Current Protocols in Immunology, Wiley lnterscience, 1991, Unit 9).Peptides of the invention can also be synthesized by the well knownsolid phase peptide synthesis methods described Merrifield, J. Am. Chem.Soc., 85:2149, 1962), and Stewart and Young, Solid Phase PeptidesSynthesis, (Freeman, San Francisco, 1969, pp.27-62), using acopoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer.On completion of chemical synthesis, the peptides can be deprotected andcleaved from the polymer by treatment with liquid HF-10% anisole forabout ¼-1 hours at 0° C. After evaporation of the reagents, the peptidesare extracted from the polymer with 1% acetic acid solution which isthen lyophilized to yield the crude material. This can normally bepurified by such techniques as gel filtration on Sephadex G-15 using 5%acetic add as a solvent. Lyophilization of appropriate fractions of thecolumn will yield the homogeneous peptide or peptide derivatives, whichcan then be characterized by such standard techniques as amino acidanalysis, thin layer chromatography, high performance liquidchromatography, ultraviolet absorption spectros-copy, molar rotation,solubility, and quantitated by the solid phase Edman degradation.

The invention also provides polynucleotides which encode the JNKpolypeptide of the invention and the synthetic peptide of SEQ ID NO: 1.As used herein, “polynucleotide” refers to a polymer ofdeoxyribonucleotides or ribonucleotides, in the form of a separatefragment or as a component of a larger construct DNA encoding thepolypeptide of the invention can be assembled from cDNA fragments orfrom oligonucleotides which provide a synthetic gene which is capable ofbeing expressed in a recombinant transcriptional unit. Polynucleotidesequences of the invention include DNA, RNA and cDNA sequences.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization procedures whichare well known in the art. These include, but are not limited to: 1)hybridization of probes to genomic or cDNA libraries to detect sharednucleotide sequences; 2) antibody screening of expression libraries todetect shared structural features and 3) synthesis by the polymerasechain reaction (PCR).

Hybridization procedures are useful for the screening of recombinantclones by using labeled mixed synthetic oligonucleotide probes whereeach probe is potentially the complete complement of a specific DNAsequence in the hybridization sample which includes a heterogeneousmixture of denatured double-stranded DNA. For such screening,hybridization is preferably performed on either single-stranded DNA ordenatured double-stranded DNA. Hybridization is particularly useful inthe detection of cDNA clones derived from sources where an extremely lowamount of mRNA sequences relating to the polypeptide of interest arepresent. In other words, by using stringent hybridization conditionsdirected to avoid non-specific binding, it is possible, for example, toallow the autoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucleic AcidResearch, 9:879, 1981).

The development of specific DNA sequences encoding JNK can also beobtained by: 1) isolation of double-stranded DNA sequences from thegenomic DNA; 2) chemical manufacture of a DNA sequence to provide thenecessary codons for the polypeptide of interest; and 3) in vitrosynthesis of a double-stranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA. Of these three methods for developingspecific DNA sequences for use in recombinant procedures, the isolationof genomic DNA isolates is the least common. This is especially truewhen it is desirable to obtain the microbial expression of mammalianpolypeptides due to the presence of introns.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the synthesis of cDNAsequences. Among the standard procedures for isolating cDNA sequences ofinterest is the formation of plasmid- or phage-carrying cDNA librarieswhich are derived from reverse transcription of mRNA which is abundantin donor cells that have a high level of genetic expression. When usedin combination with polymerase chain reaction technology, even rareexpression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay et al., Nucl. Acid Res. 11:2325, 1983).

A cDNA expression library, such as lambda gt11, can be screenedindirectly for JNK polypeptide having at least one epitope, usingantibodies specific for JNK. Such antibodies can be either polyclonallyor monoclonally derived and used to detect expression product indicativeof the presence of JNK cDNA.

A polynucleotide sequence can be deduced from the genetic code, however,the degeneracy of the code must be taken into account. Polynucleotidesof the invention include sequences which are degenerate as a result ofthe genetic code. The polynucleotides of the invention include sequencesthat are degenerate as a result of the genetic code. There are 20natural amino acids, most of which are specified by more than one codon.Therefore, as long as the amino acid sequence of JNK results in afunctional polypeptide (at least, in the case of the sensepolynucleotide strand), all degenerate nucleotide sequences are includedin the invention.

The polynucleotide sequence for JNK also includes sequencescomplementary to the polynucleotide encoding JNK (antisense sequences).Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, ScientificAmerican, 262;40, 1990). The invention embraces all antisensepolynucleotides capable of inhibiting production of JNK polypeptide. Inthe cell, the antisense nucleic acids hybridize to the correspondingmRNA, forming a double-stranded molecule. The antisense nucleic acidsinterfere with the translation of the mRNA since the cell will nottranslate a mRNA that is double-stranded. Antisense oligomers of about15 nucleotides are preferred, since they are easily synthesized and areless likely to cause problems than larger molecules when introduced intothe target JNK-producing cell. The use of antisense methods to inhibitthe translation of genes is well known in the art (Marcus-Sakura,Anal.Biochem., 172:289, 1988).

In addition, ribozyme nucleotide sequences for JNK are included in theinvention. Ribozymes are RNA molecules possessing the ability tospecifically cleave other single-stranded RNA in a manner analogous toDNA restriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer.Med.-Assn., 260;3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that that sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

The JNK polypeptides of the invention can also be used to produceantibodies which are immunoreactive or bind to epitopes of the JNKpolypeptides. Antibodies of the invention also include antibodies whichbind to the synthetic peptide in SEQ ID NO: 1. Antibody which consistsessentially of pooled monoclonal antibodies with different epitopicspecificities, as well as distinct monoclonal antibody preparations areprovided. Monoclonal antibodies are made from antigen containingfragments of the protein by methods well known in the art (Kohler, etal., Nature, 25:495, 1975; Current Protocols in Molecular Biology,Ausubel, et al., ed., 1989).

The term “antibody” as used in this invention includes intact moleculesas well as fragments thereof, such as Fab, F(ab′)_(2′), and Fv which arecapable of binding the epitopic determinant. These antibody fragmentsretain some ability to selectively bind with its antigen or receptor andare defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction;

F(ab′)₂ is a dimer of two Fab′ fragment held together by two disulfidebonds;

(4) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and

(5) Single chain antibody (“SCA”), defined as a genetically engineeredmolecule containing the variable region of the light chain, the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), incorporated herein by reference).

As used in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

Antibodies which bind to the JNK polypeptide of the invention can beprepared using an intact polypeptide or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide or apeptide such as Sequence ID No.1 used to immunize an animal can bederived from translated cDNA or chemical synthesis which can beconjugated to a carrier protein, if desired. Such commonly used carrierswhich are chemically coupled to the peptide include keyhole limpethemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanustoxoid. The coupled peptide is then used to immunize the animal (e.g., amouse, a rat, or a rabbit).

If desired, polyclonal or monoclonal antibodies can be further purified,for example, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (See for example, Coligan,et al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1991, incorporated by reference).

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody. Thus, inthe present invention, an anti-idiotype antibody produced from anantibody which binds to the synthetic peptide of the invention can bindto the site on JNK which binds to c-Jun, thereby preventing JNK frombinding to and phosphorylating c-Jun.

Polynucleotide sequences encoding the polypeptide or the syntheticpeptide (SEQ ID NO: 1) of the invention can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention.

DNA sequences encoding the polypeptides can be expressed in vitro by DNAtransfer into a suitable host cell. “Host cells” are cells in which avector can be propagated and its DNA expressed. The term also includesany progeny of the subject host cell. It is understood that all progenymay not be identical to the parental cell since there may be mutationsthat occur during replication. However, such progeny are included whenthe term “host cell” is used. Methods of stable transfer, in other wordswhen the foreign DNA is continuously maintained in the host, are knownin the art.

In the present invention, the JNK polynucleotide sequences may beinserted into a recombinant expression vector. The term “recombinantexpression vector” refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of thegenetic sequences. Such expression vectors contain a promoter sequencewhich facilitates the efficient transcription of the inserted geneticsequence of the host. The expression vector typically contains an originof replication, a promoter, as well as specific genes which allowphenotypic selection of the transformed cells. Vectors suitable for usein the present invention include, but are not limited to the T7-basedexpression vector for expression in bacteria (Rosenberg et al., Gene56:125, 1987), the pMSXND expression vector for expression in mammaliancells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein I, orpolyhedrin promoters).

The vector may include a phenotypically selectable marker to identifyhost cells which contain the expression vector. Examples of markerstypically used in prokaryotic expression vectors include antibioticresistance genes for ampicillin (β-lactamases), tetracycline andchloramphenicol (chloramphenicol acetyl-transferase). Examples of suchmarkers typically used in mammalian expression vectors include the genefor adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo,G418), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase(HPH), thymidine kinase (TK) and xanthine guaninephosphoribosyltransferse (XGPRT, gpt).

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques which are well known to those skilled in theart. Where the host is prokaryotic, such as E. coli, competent cellswhich are capable of DNA uptake can be prepared from cells harvestedafter exponential growth phase and subsequently treated by the CaCl₂method by procedures well known in the art. Alternatively, MgCl₂ or RbClcan be used. Transformation can also be performed after forming aprotoplast of the host cell or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the polypeptides of theinvention, and a second foreign DNA molecule encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein. (Eukaryotic Viral Vectors,Cold Spring Harbor Laboratory, Gluzman ed., 1982). Examples of mammalianhost cells include COS, BHK, 293, and CHO cells.

Isolation and purification of host cell expressed polypeptide, orfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

The JNK protein kinase of the invention is useful in a screening methodfor identifying compounds or compositions which affect the activity ofthe kinase. Thus, in another embodiment, the invention provides a methodfor identifying a composition which affects a c-Jun N-terminal kinasecomprising incubating the components, which include the composition tobe tested and the kinase or a polynucleotide encoding the kinase, underconditions sufficient to allow the components to interact, thensubsequently measuring the effect the composition has on kinase activityor on the polynucleotide encoding the kinase. The observed effect on thekinase may be either inhibitory or stimulatory. For example, theincrease or decrease of kinase activity can be measured by adding aradioactive compound to the mixture of components, such as ³²P-ATP, andobserving radioactive incorporation into c-Jun or other suitablesubstrate for JNK, to determine whether the compound inhibits orstimulates protein kinase activity. A polynucleotide encoding the kinasemay be inserted into an expression vector and the effect of acomposition on transcription of the kinase can be measured, for example,by Northern blot analysis.

In another embodiment, the invention provides a method of treating acell proliferative disorder associated with JNK comprising administeringto a subject with the disorder a therapeutically effective amount ofreagent which modulates kinase activity. The term “therapeuticallyeffective” means that the amount of monoclonal antibody or antisensenucleotide, for example, which is used, is of sufficient quantity toameliorate the JNK associated disorder. The term “cell-proliferativedisorder” denotes malignant as well as non-malignant cell populationswhich morphologically often appear to differ from the surroundingtissue. For example, the method may be useful in treating malignanciesof the various organ systems, such as lung, breast, lymphoid,gastrointestinal, and genito-urinary tract as well as adenocarcinomaswhich include malignancies such as most colon cancers, renal-cellcarcinoma, prostate cancer, non-small cell carcinoma of the lung, cancerof the small intestine and cancer of the esophagus.

The method is also useful in treating non-malignant orimmunological-related cell-proliferative diseases such as psoriasis,pemphigus vulgaris, Behcet's syndrome, acute respiratory distresssyndrome (ARDS), ischemic heart disease, post-dialysis syndrome,leukemia, rheumatoid arthritis, acquired immune deficiency syndrome,vasculitis, septic shock and other types of acute inflammation, andlipid histiocytosis. Especially preferred are immunopathologicaldisorders. Essentially, any disorder which is etiologically linked toJNK kinase activity would be considered susceptible to treatment.

Treatment includes administration of a reagent which modulates JNKkinase activity. The term “modulate” envisions the suppression ofexpression of JNK when it is over-expressed, or augmentation of JNKexpression when it is under-expressed. It also envisions suppression ofphosphorylation of c-Jun, for example, by using the peptide of SEQ IDNO:1 as a competitive inhibitor of the natural c-Jun binding site in acell. When a cell proliferative disorder is associated with JNKoverexpression, such suppressive reagents as antisense JNKpolynucleotide sequence or JNK binding antibody can be introduced to acell. In addition, an anti-idiotype antibody which binds to a monoclonalantibody which binds a peptide of the invention may also be used in thetherapeutic method of the invention. Alternatively, when a cellproliferative disorder is associated with underexpression or expressionof a mutant JNK polypeptide, a sense polynucleotide sequence (the DNAcoding strand) or JNK polypeptide can be introduced into the cell.

The antibodies of the invention can be administered parenterally byinjection or by gradual infusion over time. The monoclonal antibodies ofthe invention can be administered intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration of a peptide or an antibodyof the invention include sterile aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. Intravenous vehicles include fluid andnutrient replenishers, electrolyte replenishers (such as those based onRinger's dextrose), and the like. Preservatives and other additives mayalso be present such as, for example, antimicrobials, anti-oxidants,chelating agents, and inert gases and the like.

Polynucleotide sequences, including antisense sequences, can betherapeutically administered by various techniques known to those ofskill in the art. Such therapy would achieve its therapeutic effect byintroduction of the JNK polynucleotide, into cells of animals having theproliferative disorder. Delivery of JNK polynucleotide can be achievedusing free polynucleotide or a recombinant expression vector such as achimeric virus or a colloidal dispersion system. Especially preferredfor therapeutic delivery of nucleotide sequences is the use of targetedliposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such, as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. By inserting a JNK sequence into the viralvector, along with another gene which encodes the ligand for a receptoron a specific target cell, for example, the vector is now targetspecific. Retroviral vectors can be made target specific by inserting,for example, a polynucleotide encoding a sugar, a glycolipid, or aprotein. Preferred targeting is accomplished by using an antibody totarget the retroviral vector. Those of skill in the art will know of, orcan readily ascertain without undue experimentation, specificpolynucleotide sequences which can be inserted into the retroviralgenome to allow target specific delivery of the retroviral vectorcontaining the JNK polynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsitation. Helper cell lines which havedeletions of the packaging signal include but are not limited to ψ2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced. The vector virions produced by thismethod can then be used to infect a tissue cell line, such as NIH 3T3cells, to produce large quantities of chimeric retroviral virions.

Another targeted delivery system for JNK polynucleotides is a colloidaldispersion system. Colloidal dispersion systems include macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes. The preferred colloidal system of this invention is aliposome. Liposomes are artificial membrane vesicles which are useful asdelivery vehicles in vitro and in vivo. It has been shown that largeunilamellar vesicles (LUV), which range in size from 0.2-4.0 um canencapsulate a substantial percentage of an aqueous buffer containinglarge macromolecules. RNA, DNA and intact virions can be encapsulatedwithin the aqueous interior and be delivered to cells in a biologicallyactive form (Fraley, et al., Trends Biochem. Sci., 6:77, 1981). Inaddition to mammalian cells, liposomes have been used for delivery ofpolynucleotides in plant, yeast and bacterial cells. In order for aliposome to be an efficient gene transfer vehicle, the followingcharacteristics should be present: (1) encapsulation of the genes ofinterest at high efficiency while not compromising their biologicalactivity; (2) preferential and substantial binding to a target cell incomparison to non-target cells; (3) delivery of the aqueous contents ofthe vesicle to the target cell cytoplasm at high efficiency; and (4)accurate and effective expression of genetic information (Mannino, etal., Biotechniques, 6:682, 1988).

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The invention also provides a method for detecting a cell with JNKkinase activity or a cell proliferative disorder associated with JNKcomprising contacting a cell component with cJun N-terminal kinaseactivity with a reagent which binds to the component and measuring theinteraction of the reagent with the component. Such reagents can be usedto measure relative levels of JNK expression compared to normal tissue.The cell component can be nucleic acid, such as DNA or RNA, or protein.When the component is nucleic acid, the reagent is a nucleic acid probeor PCR primer. The interaction of a nucleic acid reagent with a nucleicacid encoding a polypeptide with c-Jun N-terminal kinase activity istypically measured using radioactive labels, however, other types oflabels will be known to those of skill in the art. When the cellcomponent is protein, the reagent is typically an antibody probe. Theprobes are directly or indirectly detectably labeled, for example, witha radioisotope, a fluorescent compound, a bioluminescent compound, achemiluminescent compound, a metal chelator or an enzyme. Those ofordinary skill in the art will know of other suitable labels for bindingto the antibody, or will be able to ascertain such, using routineexperimentation.

Preferably the probe for identification of a cell with JNK kinaseactivity is a c-Jun protein. JNK activity within a cell is measured bythe amount of phosphorylation of the c-Jun protein probe. For example,the amount of JNK activity in a cell extract can be measured by mixingthe extract with c-Jun protein and adding a radioactive compound, suchas ³²P-ATP to the mixture of components. The amount of radioactivitythat is incorporated into the c-Jun probe is determined, for example bySDS-PAGE, and compared to a cell control containing c-Jun and a normallevel of JNK kinase activity.

The c-Jun substrate can be immobilized onto a 96 well microtiter dishand extracts from treated cells added to the wells. The wells are thenwashed and an appropriate buffer containing ³²P-ATP is added to thewells. The phosphorylation reaction proceeds for about 15 minutes andthe wells are washed and counted. Modifications of the assay includeimmobilizing the substrate using beads or magnetic particles andnon-radioactive procedures to measure the substrate phosphorylation,such as using monoclonal antibodies and a detection system (e.g.,biotinilated antibodies and avidin peroxidase reaction).

The Jun protein used in the method of detection of the JNK kinasedescribed above may exist as a single protein unit or a fusion protein.The fusion protein preferably consists of c-Jun andglutathione-S-transferase (GST) as a carrier protein. The c-junnucleotide sequence is cloned 3′ to the carrier protein in an expressionvector, such as pGEX or such derivatives as pGEX2T or pGEX3X, the geneis expressed, the cells are lysed, and the extract is poured over acolumn containing a resin or mixed directly with a resin to which thecarrier protein binds. When GST is the carrier, a glutathione (GSH)resin is used. When maltose-binding protein (MBP) is the carrier, anamylose resin is used. Other carrier proteins and the appropriatebinding resin will be known to those of skill in the art.

The materials of the invention are ideally suited for the preparation ofa kit. The kit is useful for the detection of the level of a c-JunN-terminal kinase comprising an antibody which binds a c-Jun N-terminalkinase or a nucleic acid probe which hybridizes to JNK nucleotide, thekit comprising a carrier means being compartmentalized to receive inclose confinement therein one or more containers such as vials, tubes,and the like, each of the container means comprising one of the separateelements to be used in the assay. For example, one of the containermeans may comprise a monoclonal antibody of the invention which is, orcan be, detectably labelled. The kit may also have containers containingbuffer(s) and/or a container comprising a reporter-means (for example, abiotin-binding protein, such as avidin or streptavidin) bound to areporter molecule (for example, an enzymatic or fluorescent label).

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLE 1 Plasmids and Expression of GST Fusion Proteins

The glutathione-S-transferase (GST)-cJun expression vector,pGEX2T-cJun(wt), was constructed by inserting a filled-in BspHI-Pstlfragment (encoding AA 1-223) from RSV-cjun(BspHI) into the Smal site ofpGEX2T (Pharmacia). RSV-cJun(BspHI) was constructed by changing thetranslation initiation sequence CTATGA of RSV-cJun to TCATGA bysite-directed mutagenesis. The GSTcJun(Ala63/67)(BspHI) expressionvector was derived in the same manner from RSV-cJun(Ala63/73) (Smeal, etal., supra, 1991) and was used to construct pGEX2T-cJun(Ala 63/67). Thevarious GSTcJun truncation mutants were constructed using the polymerasechain reaction (PCR) to amplify various portions of c-Jun coding region.The sequences of the primers are indicated below:

N-terminal primers: TCTGCAGGATCCCCATGACTGCAAAGATGGAAACG (underlinedcodon: amino acid 1) (SEQ ID NO: 2);

TCTGCAGGATCCCCGACGATGCCCTCAACGCCTC (a.a. 11) (SEQ ID NO: 3);

TCTGCAGGATCCCCGAGAGCGGACCTTATGGCTAC (a.a.22) (SEQ ID NO: 4);

TCTGCAGGATCCCCGCCGACCCAGTGGGGAGCCTG (a.a. 43) (SEQ ID NO: 5),TCTGCAGGATCCCCAAGAACTCGGACCTCCTCACC (a.a 56) (SEQ ID NO: 6) C-terminalprimers: TGAATTCTGCAGGCGCTCCAGCTCGGGCGA (a.a. 79) (SEQ ID NO: 7); andTGAATTCCTGCAGGTCGGCGTGGTGGTGATGTG (a.a. 93) (SEQ ID NO: 8).

The DNA fragments were amplified by using Pfu polymerase (Strategene, LaJolla, Calif.), digested with BamHI and Pstl, and subcloned to BamHI.Pstl sites of pBluescript SK+ (Strategene). The BamHI-EcoRI fragmentswere excised from pBluescript and subcloned to BamHI, Pstl sites ofpGEX3X (Pharmacia). Some constructs were made by inserting BamHI-Avalfragments of the PCR products and the Aval-EcoRI fragment ofpGEX2T-cJun(wt) into BamHI, EcoRI sites of pGEX3X. pGEX3X-cJun(33-223)was constructed by inserting a Xholl-EcoRI fragment into pGEX3X.

The v-Jun and chick c-Jun sequences were derived from RCAS VC-3 and RCASCJ-3 respectively (Bos, et al., Genes Dev., 4:1677, 1990). GSTfusionvectors for v-Jun and chicken c-Jun were constructed by inserting Ncolfragments of RCAS VC-3 and RCAS CJ-3 into Ncol site of pGEX-KG (Guan andDixon, Anal. Biochem., 192:262, 1989). The same fragments containvarious portions of the c-Jun and v-Jun coding regions were cloned intopSG424, a GAL4 DNA binding domain expression vector (Sadowski andPtashne, Nucl. Acids Res., 17:753, 1989).

The GST fusion protein expression vectors were transformed into theXL1-Blue or NM522 strains of E. coli. Protein induction and purificationwere performed as previously described (Smith and Johnson, Gene, 67:31,1988). The amount of purified fusion protein was estimated by theBio-Rad Protein Assay Kit. In some experiments GST fusion proteins werenot eluted from the glutathione (GSH)-agarose beads and were retained onthe beads for isolation of the c-Jun N-terminal kinase.

Cell Culture and Preparation of Cell Extracts

FR3T3, Ha-ras transformed FR3T3, HeLaS3 and QT6 cells were grown inDulbecco's modified Eagle's Medium (DMEM) containing 10% fetal calfserum (FCS), 100 U/ml penicillin (Pc), and 100 μg/ml streptomycin (Sm).Jurkat, K562 and U937 cells were grown in RPMI 1640 supplemented with10% FCS, 100 U/ml Pc, and 100 μg/ml Sm. F9 cells were grown in 45% DMEM,45% Ham's F12, 10% FCS, 100 U/ml Pc and 100 μg/ml Sm. Nuclear andcytoplasmic extracts were prepared as described by Dignam, et al.,(1983). To prepare whole cell extract (WCE), harvested cells weresuspended in WCE buffer: 25 mM HEPES pH 7.7, 0.3 M NaCl; 1.5 mM MgCl₂0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM DTT, 20 mM β-glycerophosphate,0.1 mM Na₃VO₄, 2 μg/ml leupeptin, 100 μg/ml PMSF. The cell suspensionwas rotated at 4° C. for 30 minutes and the extract was cleared bycentrifugation at 10,000×g for 10 minutes. Protein amount was estimatedby Bio-Rad Protein Assay Kit.

Transfection Experiments

Transfection experiments were performed using RSV-cJun, RSV-vJun andGAL4-Jun, GAL4-vJun and Ha-Ras(Leu 61) expression vectors as previouslydescribed (Boyle, et al., supra, 1991; Binetruy, et al., supra, 1991;Smeal, et al., supra, 1991). CAT activity was determined as described inExample 8 below. c-Jun and v-Jun protein expression and phosphorylationwere analyzed as described by Smeal, et al., supra, 1991; Smeal, et al.,Mol. Cell Biol., 12:3507, 1992).

Protein Purification

GST-fusion proteins were purified by affinity chromatography onGSH-agarose as described (Smith, et al., Gene, 67:31-40, 1988). PurifiedMAP kinase (a mixture of ERK1 and ERK2) was obtained from Dr. M. Cobb(University of Texas Southwestern). JNK-46 was purified fromUV-irradiated HeLa S3 cells by standard liquid chromatography.Epitope-tagged JNK was immunopurified from transiently transfected COScells. Briefly, COS cells were solubilized with 20 mM Tris (pH 7.6),0.5% NP-40, 250 mM NaCl, 3 mM β-glycerophosphate, 3 mM EDTA, 3 mM EGTA,100 μM Na orthovanadate, 10 μg/ml leupeptin, 1 mM PMSF. JNK was isolatedby immunoaffinity chromatography using the M2 monoclonal antibody boundto protein A-Sepharose. The beads were washed extensively with Buffer A(20 mM Hepes (ph 7.7), 50 mM NaCl, 0.1 mM EDTA, 0.05% Triton X-100). JNKwas eluted from the column with 3 M urea in Buffer A and the dialyzedagainst Buffer A with 10% glycerol.

EXAMPLE 2 Kinase Assays

Solid Phase Kinase Assay

Cell extracts were diluted so that the final composition of the WCEbuffer was 20 mM HEPES pH 7.7, 75 mM NaCl, 25 mM MgCl₂, 0.1 mM EDTA,0.05% Triton X-100, 0.5 mM DTT, 20 mM β-glycerolphosphate, 0.1 mMNa₃VO₄, 2 μg/ml leupeptin, 100 μg/ml PMSF. The extracts were mixed with10 μl of GSH-agarose suspension (Sigma) to which 10 μg of either GST orGST-Jun fusion proteins were bound. The mixture was rotated at 4° C. for3 hours in a microfuge tube and pelleted by centrifugation at 10,000×gfor 20 sec. After 4×1 ml washes in HEPES binding buffer (20 mM HEPES pH7.7, 50 mM NaCl, 2.5 mM MgCl₂, 0.1 mM EDTA, 0.05% Triton X-100), thepelleted beads were resuspended in 30 μl of kinase buffer (20 mM HEPESpH 7.6, 20 mM MgCl₂, 20 mM β-glycerolphosphate, 20 μM p-nitrophenylphosphate. 0.1 mM Na₃VO₄, 2 mM DTT) containing 20 μM ATP and 5 μCiγ-³²P-ATP. After 20 minutes at 30° C. the reaction was terminated bywashing with HEPES binding buffer. Phosphorylated proteins were elutedwith 30 μl of 1.5×Laemlli sample buffer and resolved on a 10% SDSpolyacrylamide gel, followed by autoradiography. Quantitation ofphosphate incorporated was determined by gel slicing and scintillationcounting. Phosphorylated GST fusion proteins were eluted from gel slicesand subjected to phosphopeptide mapping as described (Boyle, et al.,supra, 1991).

In-Gel Kinase Assay

In-gel kinase assay was performed as described by Kameshita andFujisawa, Anal. Biochem.,183:139, (1989) with slight modifications.Briefly, c-Jun binding proteins were isolated from whole cell extractsby using GSH-agarose beads containing 80 μg GST-cJun as described above.Proteins were eluted in Laemlli sample buffer and resolved on 10%SDS-polyacrylamide gel, which was polymerized in the absence or presenceof GST-cJun (40 μg/ml). After electrophoresis, the gel was washed twice,30 minutes each time with 100 ml of 20% 2-propanol, 50 mM HEPES pH 7.6to remove SDS. After the gel was washed twice, 30 minutes each time,with 100 ml of buffer A-(50 mM HEPES pH 7.6, 5 mM β-mercaptoethanol), itwas incubated in 200 ml of 6M urea in buffer A at room temperature for 1hr, followed by serial incubations in buffer A containing 0.05% Tween 20and either 3M, 1.5M or 0.75M urea. After the gel was washed severaltimes, 1 hr each time, with 100 ml of buffer A containing 0.05% Tween 20at 4° C., it was incubated with kinase buffer containing 50 μM ATP and 5μCi/ml γ³²P-ATP at 30° C. for 1 hour. After the reaction, the gel waswashed with 100 ml of 5% tricholoroacetic acid and 1% sodiumpyrophosphate at room temperature several times, followed by drying andautoradiography.

EXAMPLE 3

BINDING OF A PROTEIN KINASE TO GST-cJun-GSH-AGAROSE BEADS

The fusion protein, GSTcJun(wt), can bind through its GST moiety toglutathione (GSH)-agarose beads to generate an affinity matrix foridentification of c-Jun binding proteins, which may include proteinkinases. Ha-ras transformation of FR3T3 cells results in increasedphosphorylation of c-Jun on Ser 63 and 73 (Binetruy, et al., supra,1991; Smeal, et al., supra, 1991). Preliminary experiments indicatedthat transformed cells contained higher levels of c-Jun N-terminalkinase activity, while the levels of c-Jun C-terminal kinase activityremained unchanged. To develop a more convenient assay forcharacterizing the c-Jun N-terminal kinase activity nuclear andcytoplasmic extracts of untransformed and transformed FR3T3 cells weremixed with GSTcJun(wt)-GSH-agarose beads. FRT3T3(−) andHa-ras-transformed FR3T3(+) cells were kept in 0.5% FCS for 24 hours andharvested to prepare nuclear and cytosolic extracts. These extracts(prepared from equal number of cells) were mixed with GSH-agarose beadscontaining 10 μg of GST-cJun(wt), GSTcJun(Ala63/73) or GST. After a 3hour incubation, the beads were spun down, washed 4-times and incubatedin kinase buffer containing β-³²P-ATP for 20 minutes at 30° C. Thereaction was terminated by washing in SDS sample buffer. The elutedproteins were resolved by SDS-PAGE. The location of the GSTcJun fusionproteins is indicated in FIG. 1. Similar results were obtained whenprotein concentration rather than cell number (300 μg of cytosolicextract and an equivalent amount of nuclear extract) was used tonormalize the amounts of extracts used in this assay. This procedureresulted in phosphorylation of GSTcJun(wt), suggesting that a proteinkinase bound to it and phosphorylated it while attached to GSH-agarose(FIG. 1). On the other hand, no phosphorylation of GST bound toGSH-agarose could be detected by this assay.

The same experiment was repeated using a GSTcJun(Ala63/73) fusionprotein, in which both the serine at position 63 and 73 were convertedto alanines in order to identify a kinase that targets Ser 63 and 73 ofc-Jun. Phosphorylation of this protein was considerably lower than thatof GSTcJun(wt) (FIG. 1). These experiments confirmed the previousobservations that the kinase activity affecting the N-terminal sites ofc-Jun was elevated upon Has-ras transformation and are consistent withthe differences in the extent of c-Jun N-terminal phosphorylationbetween transformed and untransformed cells detected by in vivolabelling (Binetruy, et al., supra, 1991; Smeal, et al., supra, 1991,1992). The kinase activity detected by this solid-phase assay waspresent in both the cytosolic and the nuclear fractions and wasseveral-fold more abundant in the cytosol on a per-cell basis. However,it is possible that some of the kinase leaked from the nuclei to thecytosol during the cell fractionation.

The solid-phase assay was used to examine N-terminal c-Jun kinaseactivity in other cell types. Exposure of HeLa cells to UV activates theHa-Ras signalling pathway and results in a large increase in N-terminalphosphorylation of c-Jun (Devary, et al., Cell, 71:1081, 1992).Treatment of HeLa cells with the phorbol ester, TPA, on the other hand,has only a marginal effect on N-terminal phosphorylation of c-Jun(Boyle, et al., 1991). HeLa S3 cells were serum starved for 12 hours andwere either left untreated, irradiated with UV light (40 J/m²) orincubated with TPA (100 ng/ml). The cells were harvested at theindicated times (min) after UV or TPA exposure. Whole cell extracts(approximately 800 μg protein) isolated form equal numbers of cells weremixed with GSH-agarose beads containing 10 μg of either GST,GSTcJun(wt), or GSTcJun(Ala 63/73). After 3 hours incubation, followedby extensive washing, the solid state phosphorylation assay wasperformed as described above. After a 20 minute reaction, the proteinswere dissociated in SDS sample buffer and resolved by SDS-PAGE.

As shown in FIG. 2A, N-terminal c-Jun kinase activity was elevatedwithin 5 minutes after UV irradiation and was 250-fold higher after 30minutes than in unstimulated cells. The effect of TPA, however, wasminor compared to that of UV. As found before, GSTcJun(wt) was moreefficiently phosphorylated than GSTcJun(Ala63/73), whereas GST was notphosphorylated. These results are consistent with in vivo measurementsof c-Jun phosphorylation (Boyle, et al., supra, 1991; Devary, et al.,supra, 1992).

TPA treatment of Jurkat T cells, in contrast to HeLa cells, resulted instimulation of c-Jun phosphorylation on Ser 63 and 73. Jurkat cells wereserum starved for 2 hours and either left untreated or stimulated withTPA (50 ng/ml) for 10 or 30 minutes. Whole cell extracts prepared from5×10⁶ cells were mixed with GSH-agarose beads containing GST,GSTcJun(wt) or GSTcJun(Ala63/73). Phosphorylation of the GST proteinsattached to the beads was performed as described above. The fastermoving bands correspond to degradation products of the GSTcJun proteins.

In Jurkat cells, unlike HeLa cells, the N-terminal kinase activity wasfound to be strongly activated by TPA (25-fold after 30 minutes) (FIG.2B). This kinase also preferred GSTcJun(wt) over GSTcJun(Ala63/73) anddid not bind to or phosphorylate the GST moiety. Collectively, thesefindings suggest that the kinase detected by the solid-phase assayphosphorylates c-Jun on Ser 63 and 73 and that its regulation parallelsthat of c-Jun N-terminal phosphorylation examined by in vivo labelling.

EXAMPLE 4 Phosphorylation of Serines 63 and 73 by Bound Kinase, JNK

To determine the exact phosphoacceptor sites used by the kinase thatbinds to GSTcJun, the phosphorylated GSTcJun(wt) and GSTcJun(Ala63/73)proteins were subjected to two-dimensional tryptic phosphopeptidemapping. Whole cell extracts of Ha-ras-transformed FR3T3 cells (2.5 mg),UV irradiated HeLa cells (200 μg) or TPA-stimulated Jurkat cells (1.2mg) were mixed with GSH-agarose beads, containing either GSTcJun(wt) orGSTcJun(Ala63/73). The GSTcJun proteins were phosphorylated as describedabove by the bound kinase, isolated by SDS-PAGE, excised from the gel,digested with trypsin and subjected to two-dimensional phosphopeptidemapping. The X, Y, T1, and T2 phosphopeptides are indicated. All theautoradiograms were exposed for the same length of time.

As shown in FIG. 3A, the kinases isolated from Ha-ras-transformed FR3T3cells, UV-irradiated HeLa cells and TPA-stimulated Jurkat cells,phosphorylated GSTcJun on X, Y, and two other peptides, T1 and T2. X andY correspond to phosphorylation of Ser-73 -and Ser-63, respectively(Smeal, et al., supra, 1991) and were absent in digests ofGSTcJun(A1a63/73), which contained higher relative levels of T1 and T2.Phosphoaminoacid analysis indicated that T1 and T2 contain onlyphosphothreonine. By deletion analysis these threonines were assigned toAA 91, 93 or 95 of c-Jun.

As described below, the kinase bound to GSTcJun was eluted from thebeads and used to phosphorylate recombinant full-length c-Jun protein insolution (FIG. 3B). Recombinant c-Jun protein was phosphorylated invitro by the c-Jun N-terminal kinase (JNK) eluted fromGSTcJun(WT)-GSH-agarose beads. In addition, c-Jun was isolated byimmuneprecipitation from ³²P-labelled F9 cells that were cotransfectedwith c-Jun and Ha-Ras expression vectors (Smeal, et al, supra, 1991).Equal counts of each protein preparation were digested with trypsin andsubjected to phosphopeptide mapping. The migration positions of the X,X′ (a derivative of X generated by alkylation; Smeal, et al., supra,1991) Y, b and c phosphopeptides are indicated.

As found in vivo, the bound kinase phosphorylated c-Jun mostly on Ser73, followed by phosphorylation of Ser 63. In addition, the bound kinaseactivity phosphorylated c-Jun weakly on two of its C-terminal sites,resulting in appearance of phosphopeptides b and c. Since this is thefirst protein kinase that was detected with clear specificity for atleast one of the N-terminal sites of c-Jun, it was named JNK, for cJunN-terminal protein-kinase.

EXAMPLE 5 Binding of JNK to cJun

To examine the stability of the interaction between GSTcJun and JNK,extracts of TPA-stimulated Jurkat cells were incubated withGSTcJun(wt)-GSH-agarose beads. After extensive washing, the beads weresubjected to elution with increasing concentrations of NaCl, urea,guanidine-HCl and SDS. Elution of JNK was examined by its ability tophosphorylate recombinant c-Jun in solution. GSTcJun(wt)-GSH-agarosebeads were incubated for 3 hours with a whole cell extract ofTPA-stimulated Jurkat cells and after four washes were subjected toelution in kinase buffer containing increasing concentrations of NaCl,urea, guanidine-HCl (in M) or SDS (in %) (FIG. 4). The eluted fractions(equal volumes) were dialyzed at 40° C. against kinase buffer containing10% glycerol and no ATP and then incubated with recombinant c-Junprotein (250 ng) in the presence of 20 μM ATP and 5 μCi of γ-³²P-ATP for20 minutes at 30° C. The amount of kinase remaining on the beads afterthe elution steps (R lanes) was determined by incubation of the isolatedbeads with kinase buffer in the presence of 20 μM ATP and 5 μCiγ-³²P-ATP for 20 minutes at 30° C. The phosphorylated proteins wereanalyzed by SDS-PAGE as described above and visualized byautoradiography. The migration positions of GSTcJun and, c-Jun areindicated.

Surprisingly, JNK was found to bind GSTcJun rather tightly; only a smallfraction of kinase activity was eluted by 0.5M NaCl and even afterelution with 2M NaCl, most of the kinase remained on the beads (FIG.4A). Approximately 50% of the bound kinase was eluted by 1M urea and therest was eluted by 2M urea. Nearly complete elution was achieved byeither 0.5M guanidine-HCl or 0.01% SDS. Under all of these elutionconditions, GSTcJun(wt) was also partially eluted from the GSH-agarosebeads. This suggests that the stability of the JNK:c-Jun complex issimilar to that of the GST:GSH complex.

GSTcJun(wt) was covalently linked to GSH-agarose beads, usingcyanogen-bromide, and incubated with a whole cell extract ofTPA-stimulated Jurkat cells. After extensive washing, part of the beadswere eluted with kinase buffer-containing: no ATP (FIG. 4B, lane 2), 20μM ATP (lane 3) or 50 μM ATP (lane 4). The eluted fractions (equalvolumes) were incubated with recombinant c-Jun protein (500 ng) as asubstrate and 5 μCi γ-³²P-ATP for 30 minutes. In addition, the beadsafter elution with either kinase buffer alone (lane 1) or kinase buffercontaining 50 μM ATP (lane 5) were incubated with c-Jun protein (500 ng)in the presence of 5 μCi γ-³²P-ATP for 30 minutes. Phosphorylation ofc-Jun (indicted by the arrow) was analyzed by SDS-PAGE andautoradiography.

Addition of exogenous c-Jun to kinase-loaded GSH-agarose beads to whichGSTcJun was covalently linked results in its efficient phosphorylation(FIG. 4B, Lane 1). This suggests that after phosphorylating GSTcJun, JNKdissociates from it and phosphorylates exogenous c-Jun. In addition,incubation with kinase buffer containing ATP resulted in elution of JNKfrom the GSTcJun beads, as indicated by its ability to phosphorylateexogenous c-Jun (FIG. 4B, lanes 2-4). After incubation with 50 μM ATPless than 20% of the kinase remained on the beads (compare lanes 1 and5, FIG. 4B).

EXAMPLE 6 JNK1 is a 46 kD Protein

An in-gel kinase assay was performed to determine the size of JNKGSTcJun-GSH-agarose beads were incubated with a whole cell extract ofTPA-stimulated Jurkat cells, washed extensively and the bound proteinswere eluted in SDS sample buffer and separated on SDS-polyacrylamidegels that were polymerized in the absence (−) or presence (+) ofGSTcJun(wt). After electrophoresis, the gel was incubated in 6 M ureaand subjected to renaturation as described in Example 1. The renaturedgels were incubated in kinase buffer containing 50 μM ATP and 5 μCi/mlγ-³²P-ATP for 1 hour at 30° C., washed, fixed, and visualized byautoradiography.

In both cases a protein band whose apparent molecular weight was 46 kDwas phosphorylated (FIG. 5A). Phosphorylation was 2-fold more efficientin the presence of GSTcJun. This indicates that 46 kD protein band iseither autophosphorylated JNK or a comigrating protein. No ³²P-labelledprotein was detected in eluates of GST-GSH-agarose beads.

The same in-gel kinase assay was used to demonstrate increased JNKactivity upon TPA stimulation of Jurkat cells or UV irradiation of HeLacells (FIG. 5B). GSTcJun-GSH-agarose beads were incubated with wholecell extracts of unstimulated or UV-stimulated HeLa cells andunstimulated or TPA-stimulated Jurkat cells. After washing, the boundproteins were eluted in SDS sample buffer and separated by SDS-PAGEAfter renaturation, the gel was incubated in kinase buffer containing 50μM ATP and 5 μCi/ml γ-³²P-ATP and the phosphorylated proteins werevisualized by autoradiography.

These results provide further evidence that the apparent molecularweight of JNK is 46 kD. To determine whether the same N-terminal c-Junkinase is present in various cell types, the in-gel kinase assay wasused to examine extracts of K562 human erythroleukemia cells, U937 humanhistiocytic leukemia cells, Jurkat cells, HeLa cells, F9 embryonalcarcinoma cells, Ha-ras-transformed FR3T3 cells and QT6 quailfibroblasts. The HeLa, F9 and QT6 extracts were prepared formUV-irradiated cells and the U937 and Jurkat extracts were made fromTPA-stimulated cells, while the K562 cells were not subjected to anyspecial treatment. All cells contained a protein kinase that bound toGSTcJun and migrated around 46 kD (FIG. 5C). Some cells, especially QT6cells, contained a second less abundant protein kinase species,migrating at about 55 kD. The activities of both kinases were induced bycell stimulation. GSTcJun(WT)-GSH-agarose beads were incubated withwhole cell extracts of logarithmically growing K562 and Ha-rastransformed FR3T3 cells, TPA-stimulated Jurkat and U937 cells andUV-irradiated HeLa, F9 and QT6 cells. After washing, the bound proteinswere eluted and analyzed by in-gel kinase assay as described above.

Further evidence that JNK is 46 kD in size was obtained by separatingthe GSTcJun-bound protein fraction of TPA-stimulated Jurkat cell extractby SDS-PAGE. After elution and renaturation of the fractionatedproteins, the molecular weight of the major protein kinase bound toGSTcJun, capable of specific phosphorylation of Ser 63 and 73, wasdetermined to be 46 kD. Although the sizes of ERK1 and ERK2, 44 and 42kD, respectively, are close to that of JNK. Western blot analysis, usingan antiserum that reacts with both ERK's, indicates that the 46 kD JNKis not immunologically related to either of them. In addition, JNK isnot immunologically related to Raf-1. In addition, a 55 kD polypeptidewas identified as exhibiting JNK activity, however, the 46 kD appears tobind c-Jun more efficiently (Hibi, et al., Genes Dev., 7:2135, 1993).

EXAMPLE 7 Delineation of the Kinase Binding Site

Deletion mutants of GSTcJun lacking either N-terminal or C-terminalsequences (FIG. 6A) were used to define the JNK binding site. GSTcJunfusion proteins containing various c-Jun sequences were expressed in E.coli and isolated by binding to GSH-agarose. The bound proteins wereanalyzed by SDS-PAGE and stained with Coomassie Blue. Numbers indicatethe amino acids of c-Jun present in each fusion protein. The migrationpositions of the intact GST-fusion proteins are indicated by the dots.Faster migrating bands are degradation products.

These proteins were immobilized on GSH-agarose beads and incubated withan extract of UV-irradiated HeLa cells. Whole cell extracts ofUV-irradiated HeLa S3 cells were mixed with GSH-agarose beads containingequal amounts of the various GST fusion proteins. After washing, thebeads were incubated for 20 minutes in kinase buffer containingγ³²P-ATP. The GST fusion proteins were eluted from the beads andanalyzed by SDS-PAGE and autdradiography. The migration positions of theintact GST fusion proteins are indicated by the dots. After incubationwith whole cell extracts of UV-irradiated HeLa cells and washing, partof the bound JNK fraction was eluted with 1 M NaCl and examined for itsability to phosphorylate recombinant cJun (250 ng) in solution. Proteinphosphorylation was analyzed by SDS-PAGE and autoradiography.

Binding of JNK was examined by its ability to phosphorylate the GSTcJunfusion proteins, all of which contained both Ser 63 and 73 (FIG. 6B). Toexclude the possibility that any of the truncations may have altered theconformation of c-Jun affecting the presentation of its N-terminalphosphoacceptors without affecting JNK binding, the kinase eluted fromthese beads was examined for its ability to phosphorylate exogenousfull-length c-Jun in solution (FIG. 6C). The results obtained by bothassays indicated that removal of amino acids (AA) 1-21 had no effect onJNK binding. Removal of AA 1-32 decreased phosphorylation of GSTcJun buthad only a small effect on kinase binding. Removal of AA 1-42 however,completely eliminated kinase binding. In contrast to the N-terminaltruncations, the two C-terminal truncations, that were examined, had noeffect on JNK binding and a GST fusion protein containing AA 1-79 ofc-Jun exhibited full binding activity. Hence, AA 33-79 constitute thekinase binding site.

The JNK binding site encompasses the 6 region, spanning AA 31-57 ofc-Jun that are deleted in v-Jun (Vogt and Bos, 1990). To determine theinvolvement of the 6 region in kinase binding, GST fusion proteinscontaining the N-terminal activation domain of chicken c-Jun (AA 1-144),or the equivalent region of v-Jun (FIG. 7A) were constructed. Theactivation domain (AA 1-144) of chicken (ch) c-Jun and the equivalentregion of v-Jun were fused to GST and expressed in E. coli. GST fusionproteins were isolated on GSH-agarose beads and analyzed by SDS-PAGE andCoomassie Blue staining. The migration positions of the intact proteinsare indicated by the dots. After loading these GST fusion proteins ontoGSH-agarose the kinase binding assays were performed as described above.

Extracts of TPA-activated Jurkat cells were incubated with GSH-agarosebeads containing GST, GSTcJun(Ch) or GSTcJun. After washing, the beadswere incubated in kinase buffer containing γ-³²P-ATP and thephosphorylated GST fusion protein were analyzed as described for FIG. 6.The bound protein fraction was eluted from the GSTcJun(Ch) and GSTcJunbeads and analyzed for its ability to phosphorylate c-Jun in solution,as described for FIG. 6. While chicken GSTcJun bound the kinase asefficiently as human GSTcJun, GSTcJun was defective in-JNK binding (FIG.7B, C).

EXAMPLE 8 JNK Binding is Required for Ha-ras and UV Responsiveness

Phosphorylation of Ser 63 and 73 is necessary for potentiation of c-Junmediated transactivation by Ha-Ras (Smeal, et al., supra, 1991). Ifbinding of JNK has any role in this response, mutations that decreasekinase binding in vitro should attenuate the stimulation of c-Junactivity by Ha-Ras in vivo. This relationship was examined bycotransfection assays. Expression vectors were constructed to expresschimeric GAL4-cJun and GAL4-vJun proteins, that consist of the DNAbinding domain of the yeast activator GAL4 (Sadowski and Ptashne, 1989)and N-terminal sequences of c-Jun or v-Jun. The ability of thesechimeras to activate the GAL4-dependent reporter 5×GAL4-Elb-CAT (Lillieand Green, 1989) was examined in the absence or presence of acotransfected Ha-Ras expression vector (FIG. 8A). F9 cells werecotransfected with 1.0 μg of expression vector encoding the indicatedGAL4-cJun chimeric proteins containing various portions of the c-Junactivation domain [cJ=AA1-223; 33-AA33-223; 56=AA56-223; A63,73=AA1-246(Ala63/73)] and 2.0 μg of a 5×GAL4-Elb-CAT reporter in theabsence or presence of the indicated amounts (in μg) ofpZIPNeoRas(Leu61). The total amount of expression vector was keptconstant and the total amount of transfected DNA was brought to 15 μgusing pUC18 and the appropriate amount of pZIPneo. Cells were harvested28 hours after transfection and CAT activity was determined. Shown arethe averages of two experiments, calculated as fold-activation over thelevel of reporter expression seen in the absence of the GAL4-Junexpressions vectors.

While deletion of AA 1-32 of c-Jun resulted in a small decrease inHa-Ras responsiveness (9.8-fold induction vs. 19-fold induction for wtGAL4-cJun), deletion of M 1-42 or 1-55 resulted in a greater decrease inHa-Ras responsiveness (5.2-fold induction). A similar decrease in Ha-Rasresponsiveness was observed upon substitution of c-Jun sequences withv-Jun sequences (4.7-fold induction). In fact, the GAL4-cJun(56-223) andGAL4-vJun chimeras were only 2-fold more responsive thanGAL4-cJun(1-246;Ala63/73) in which Ser 63 and 73 were converted toalanines. That chimera exhibited only a marginal response (2-fold) toHa-Ras. The same set of GAL4-cJun and GAL4-vJun fusion proteins wastested for UV responsiveness. F9 cells were transfected as describedabove except that instead of cotransfection with pZIPNeoRas, the cellswere either exposed or not exposed to 40 J/m2 of UV-C 8 hours aftertransfection. The cells were harvested and assayed for CAT activity 20hours later. FIG. 8B shows the averages of two experiments calculated asdescribed above.

As shown in FIG. 8B, those proteins incapable of binding JNK in vitro,were non-responsive to UV in vivo. While the activity ofGAL4-cJun(1-223) was stimulated 7.5-fold by UV, the activities ofGAL4-cJun(43-223). GAL4-cJun(56-223) and GAL4-vJun were induced only1.5-fold.

To reveal the role of JNK binding in c-Jun phosphorylation, F9 cellswere transfected with c-Jun and v-Jun expression vectors in the absenceor presence of an activated Ha-Ras expression vector. UV-irradiation wasalso used to activate the Ha-Ras pathway (Devary, et al., 1992). v-Junand c-Jun were isolated by immunoprecipitation from ³²S- or ³²P-labelledF9 cells that were transfected with v-Jun and c-Jun expression vectorsin the absence or presence of pZIPNeoRas (Leu61). The isolated proteinswere analyzed by SDS-PAGE and autoradiography. Shown are the results ofone typical experiment for each protein. Note that the ³²P-labelledv-Jun autoradiogram was exposed 3 times longer than the correspondingc-Jun autoradiogram to generate signals of similar intensity. v-Jun andc-Jun were isolated from ³²P and ³²S-labelled F9 cells that weretransfected with v-Jun or c-Jun expression vectors. One half of thecells were irradiated with UV-C(40 J/m²) for 30 minutes prior toisolation of the JuR proteins by immunoprecipitation. In this case, thec-Jun and v-Jun lanes represent equal autoradiographic exposures. Thetwo arrowheads indicate the migration positions of the two forms ofc-Jun (Devary, et al., 1992), whereas the square indicates the migrationposition of v-Jun.

Immunoprecipitation from ³²S-labelled cells showed that c-Jun and v-Junwere expressed at similar levels and that their expression level was notaffected by either Ha-Ras (FIG. 9A) or UV (FIG. 9B). Immunoprecipitationfrom ³²P-labeled cells indicated that both Ha-Ras and UV stimulated thephosphorylation of c-Jun, whereas the phosphorylation of v-Jun, whosebasal level was several-fold lower than that of c-Jun, was not enhancedby either treatment. As observed previously (Devary, et al., supra,1991), UV was a stronger inducer of c-Jun phosphorylation resulting inits retarded electrophoretic mobility. Phosphopeptide mapping confirmedthat Ha-Ras expression had a much smaller effect on the phosphorylationof v-Jun in comparison to its effect on c-Jun. As shown previously(Smeal, et al., supra, 1991), v-Jun was phosphorylated only on one sitewhich is equivalent to Ser 73 of c-Jun.

EXAMPLE 9 Antisera and Proteins

c-Jun polyclonal antiserum was described by Binetruy, et al., (Nature,35:122-127, 1991). The anti-CD3 monoclonal antibody OKT3 (Van Wauwe, etal., J. Immunol., 124:2708-2713, 1980) was obtained from Dr. AmnonAltman, La Jolla Institute for Allergy and Immunology, and the anti-CD28monoclonal antibody 9.3 is described in Hansen, et al., (Immunogenetics,10:247-260, 1980). The anti-ERK2 and ant-ERK antibodies were provided byDrs. M. Weber and M. Cobb (University of Texas Southwestern),respectively. Expression and purification of GST-cJun(1-223) wasdescribed (Hibi, et al., Genes & Dev., 7:2135, 1993). The bacterialexpression vector for kinase-defective ERK-1 was a gift from Dr. M. Cobband the recombinant protein was prepared and purified by Dr. J.Hagstrom. MBP was purchased from Sigma.

Cell Culture, Metabolic Labeling, and Immunoprecipitation

Jurkat cells were grown in RPMI with 10% fetal calf serum (FCS), 1 mMglutamate, 100 μ/ml penicillin (pen), 100 μg/ml streptomycin (strep) and250 ng/ml amphotericin (complete medium). HeLa S3, CV-1 and FR3T3 cellswere grown in DMEM supplemented with 10% FCS, 100 μ/ml pen, 100 μg/mlstrep. All cells were cultured at 37° C. with 5% CO₂. Mouse thymocyteswere prepared from 8 week old Balb/C mice by gradient centrifugation onlymphocyte separation medium (Pharmacia). The lymphocytes were culturedfor 5 hours at 37° C. in RPMI+10% FCS, prior to stimulation. Jurkatcells were labelled for 90 minutes with 0.5 mCi/ml ³²P-orthophosphate(ICN Radiochemicals) in medium lacking sodium phosphate. Labelled cellswere treated with TPA (Sigma) and A23187 (Calbiochem) 1 μg/ml asindicated. When used, cyclosporin A (CsA) (Sandoz) 100 ng/ml in ethanolwas added 10 minutes prior to cell stimulation. Following stimulation,the labelled cells were washed twice with ice-cold PBS then lysed withRIPA buffer (10 mM Tris pH 7.5, 150 mM NaCl, 2 mM. EDTA, 1% Triton-X100, 1% DOC, 0.1% SDS) supplemented with phosphatase inhibitors (20 mMβ-glycerophosphate, 10 mM p-nitro-phenylphosphate, 1 mM Na₃ VO₄), andprotease inhibitors (10 μg/ml leupeptin, aprotonin, pepstatin and 1 mMphenytmethyl sulfonylfluoride). c-Jun was immunoprecipitated asdescribed (Binetruy, et al., supra., 1992) and analyzed by SDS-PAGE,followed by peptide mapping (Boyle, et al., Cell, 64:573-584, 1991; Lin,et al. Cell, 70;777-789, 1992). Ha-Ras was immunoprecipitated withY13-259. Ha-Ras bound nucleotides were extracted and analyzed asdescribed by Satoh, et al., (Proc. Natl. Acad. Sci., USA, 15:5993-5997,1990).

RNA Extraction and Northern Blot Analysis

Exponentially growing Jurkat cells (10⁶/ml) grown in complete RPMImedium was pretreated with CsA for 15 minutes when applicable, thensubjected to various treatments for another 40 minutes. Totalcytoplasmic RNA was extracted as previously described (Angel, et al.,Cell. 4:729-739, 1987). 10 μg RNA was denatured by incubating withglyoxal for 60 minutes at 55° C. and fractionated on a 1% agarose gel inphosphate buffer. The fractionated RNA was blotted to Zetabind Nylonmembrane (CUNO Labs) and hybridized to ³²P-labelled cDNA probes specificfor c-jun, jun-B, jun-D, c-fos, α-tubulin and IL-2.

Protein Kinase Assays

Exponentially growing cells were stimulated for the indicated times andhypotonic detergent cellular extracts were prepared as described (Hibi,et al., Genes and Dev., supra, 1993). The solid-state phosphorylationassay for measuring JNK activity was performed by incubated extractswith GSTcJun(1-223)-GSH agarose beads as described (Hibi, et al.,supra., 1993) and as in Example 2 ERK1 and 2 activity was assayed by animmunecomplex kinase assay using MBP as a substrate (Minden, et al.,Nature, 1993).

Reporters, Expression Vectors and Transfections

−79 jun-LUC, −73/+63 Col-LUC, −60/+63 Col-LUC were described previously(Deng and Karin, 1993). The IL2-LUC reporter plasmid was constructed bysubcloning the IL-2 promoter (298 bp) from IL2CAT/+1 (Serfling, et al.,EMBO J., 8:465-473, 1988) into the p20Luc vector (Deng and Karin, Genesand Dev., 7:479, 1993) between the Sacl and Kpnl site. The c-Junexpression vector pSRallc-Jun was constructed by subcloning the humanc-Jun HindIII-Notl fragment from pRSVc-Jun (Binetruy, et al., supra.,1991) into pSRαll vector by blunt end ligation. pBJ-CNA and pBJ-CNB werefrom Or. G. Crabtree, Stanford University. β-Actin-LUC was from Dr. C.Glass, UCSD. T Ag Jurkat cells, a derivative of the human Jurkat T-cellline stably transfected with the SV40 large T antigen (a gift from Dr.G. Crabtree) were grown to 10⁶/ml, then resuspended at 2×10⁷/ml in freshcomplete medium. 10⁷ cells (0.5 ml) were mixed with reporter plasmids (5μg, −79 jun-LUC; 10 μg, −73/+63 Col-LUC or −60/+63 Col-LUC; 5 μgIL2-LUC) at room temperature for 10 minutes, then electroporated at 250V, 960 uF in a 0.4 cm cuvette using a Bio-Rad GenePulser. Afterelectroporation, cells were immediately put on ice for 10 minutes, thenresuspended in 10 ml complete medium for 24 hours before stimulation.0.5 μg of pSRallc-Jun were used to transfect 10⁷ Jurkat cells.Luciferase activity was determined as described (Deng and Karin, supra.,1993).

Analysis of GDP and GTP Bound to RAS p21

Jurkat cells 10×10⁶ were labelled for 3 hours with ³²P-orthophosphate(ICN Radiochemicals) at 1 mCi/ml in 5 mM of Na₃VO₄ phosphate-free DMEMsupplemented with 1 mg/ml BSA. Before harvest, cells were stimulatedwith TPA, 10 ng/ml, A23187, 1 μg/ml anti-CD3 antibody (OKT3), 10 μg/ml,anti-CD28 antibody, 2 μg/ml or their combinations. After treatment for aspecified period, cells were washed once immediately with ice cold PBS,twice with ice-cold Tris-Buffered saline (50 mM Tris-HCl, pH 7.5, 20 mMMgCl₂, 150 mM NaCl, 10.5% Nonidet P-40/1 μg/ml of aprotinin, leupeptin,pepstatin and 1 mM phenylmethyl sulfonylfluoride). Ras p21 wasimmunopreciptated with monoclonal antibody Y 12-259 (Santa CruzBiotechnology, Inc., Santa Cruz, Calif.). The GDP/GTP content of Ras wasanalyzed by TLC as described (Satoh, et al., Proc. Natl. Acad. Sci.,USA, 15:5993, 1990) and quantitated with an Ambis radioanalytic imagesystem (Ambis, San Diego, Calif.).

EXAMPLE 10 Synergistic Induction of AP-1 Activity During T CellActivation

During the first stage of T lymphocyte activation, early response genesare rapidly incuded (Crabtree, Science, 243:355,361, 1989; Zipfel, etal., Mol. Cell Bio., 9:1041-1048, 1989). Induction of jun and fos genesduring activation of the Jurkat T cell line was investigated. Twodifferent co-stimulatory paradigms were used, one employing TPA and theCa²⁺ ionophore A23187, and the second based on simultaneous stimulationof the TCR complex with an antibody to its CD3 component (OKT3; VanWauwe, et al., J. Immunol., 124:2708-2713, 1980) and stimulation of theCD28 auxiliary receptor with an anti-CD28 antibody (9.3; Hansen, et al.,Immunogenetics, 10:247-260, 1993; June, et al., Immunol. Today,11:211-216, 1990). Total cytoplasmic RNA was extracted from Jurkat cellsthat were incubated with 50 ng/ml TPA (T), 1 μg/ml A23187 (A) or 100ng/ml cyclosporin A (CsA) for 40 minutes, either alone or incombinations, as indicated. After fractionation of 10 μg samples on anagarose gel and transfer to nylon membrane, the level of c-jun, jun-B,jun-D, c-fos and α-tubulin expression was determined by hybridization torandom primed cDNA probes.

Second, Jurkat cells were incubated with 10 μg/ml soluble anti-CD3(OKT3), 2 μg/ml soluble anti-CD28 (9.3) or a combination of 50 ng/ml TPAand 1 μg/ml A23817 (T/A) as indicated for 40 minutes. Total cytoplasmicRNA was isolated and 10 μg samples were analyzed as described aboveusing c-jun, jun-D and c-fos probes. IL-2 induction by the sametreatments was measured after 6 hours of stimulation by blothybridization using IL-2 and α-tubulin specific probes.

Both the first and second costimulatory paradigms induced IL-2transcrption (FIG. 11B). Optimal induction of c-jun also required acombined treatment with TPA and A23187 (FIG. 11A) or anti-CD3 andanti-CD28 (FIG. 11B). The synergistic induction of c-jun by bothcostimulatory paradigms was partially inhibited by CsA. jun-B was alsoinduced by TPA, but its induction was not affected by A23187 or CsA.Although TPA+A23187 potentiated jun-D expression, this effect was alsonot inhibited by CsA. As reported by Matilla, et al., (EMBO J.,9:4425-4433, 1990), maximal induction of c-fos also required treatmentwith TPA+A23187, but was not inhibited by CsA. Therefore sensitivity toCsA is unique to c-jun. While incubation with soluble anti-CD3 led toinduction of c-jun and c-fos, only c-jun expression was augmented bysimultaneous exposure to anti-CD28.

The effects of the different stimuli on AP-1 transcriptional activity inJurkat cells were examined using a truncated, AP-1 responsive, humancollagenase promoter (Angel, et al., Cell, 49:729-739, 1987) fused tothe luciferase (LUC) reporter gene. Jurkat cells were transfected with10 μg of either −73Col-LUC or −60Col-LUC reporter plasmids. 24 hoursafter transfection, the cells were aliquoted into 24 well plates andincubated for 9 hours with 50 ng/ml TPA, 1 μg/ml A23187 or 100 ng/mlCsA, either alone or in combinations, as indicated. The cells wereharvested and luciferase activity was determined. The results shown areaverages of three experiments done in triplicates.

While TPA and A23187 administered alone had marginal effects on−73Col-LUC, the two together resulted in its synergistic activation(FIG. 11C). The −60Col-LUC reporter, lacking an AP-1 binding site, wasnot induced. Induction of −73Col-LUC was inhibited by CsA. Treatmentwith anti-CD3 and anti-CD28 also resulted in synergistic activation of−73Col-LUC. Similar results were obtained with the AP-1 responsive c-junpromoter. These findings differ from previous measurements of AP-1activity in Jurkat cells that relied on the use of synthetic promoterscontaining multiple AP-1 sites (Matilla, et al., supra, 1993; Ullman, etal., Genes & Dev., 7:188-196, 1993). While these findings werereproducible, previous studies indicate that the physiologicalcollagenase and c-jun promoters provide a more accurate and validmeasurement of AP-1 transcriptional activity. Indeed, the expressionpatterns of the collagenase and c-jun reporters are very similar to thatof the c-jun gene.

EXAMPLE 11 Costimulation of c-Jun N-Terminal Phosphorylation isSuppressed by CsA

Induction of c-Jun transcription and optimal stimulation of AP-1correlate with changes in c-Jun phosphorylation (Devary, et al., Cell,71:1081-1091, 1992). The effect of TPA and A23187 on c-Junphosphorylation in Jurkat cells was examined. To elevate c-Junexpression, Jurkat cells were transfected with a c-Jun expressionvector. The cells were labelled with ³²P and c-Jun wasimmunoprecipitated from cells subjected to various stimuli and analyzedby SDS-PAGE (FIG. 12A). Jurkat cells (10⁶ cells per lane) weretransfected with 0.5 ug of a SRα-cJun expression vector and 24 hourslater were labeled for 3 hours with ³²P-orthophosphate (1 mCi/ml). After15 minutes, treatment with 50 ng/ml TPA (T) 1 μg/ml A23187 (A) and 100ng/ml. CsA, either alone or in combination, as indicated, the cells werelysed in RIPA buffer and c-Jun was isolated by immunoprecipitation andanalyzed by SDS-PAGE The c-Jun bands are indicated.

In unstimulated cells, phosphorylated c-Jun migrated as a single band.Treatment with TPA for 15 minutes induced the appearance of slowermigrating bands and costimulation with A23187 enhanced this effect,while CsA reduced the Ca⁺⁺ effect. Within the short time frame of thisexperiment, there were minimal effects on c-Jun expression.

Similar results were obtained by analysis of endogenous c-Jun expressionand phosphorylation (FIG. 12B). 2×10⁷ Jurkat cells were labeled for 3hours with either ³⁵S-methionine (900 μCi/ml) or ³²P-orthophosphate (1mCi/ml). After 15 minutes incubation with 50 ng/ml TPA+1 μg/ml A23178(T/A) in the absence or presence of and 100 ng/ml CsA or no addition, asindicated, the cells were lysed in RIPA buffer and c-Jun isolated byimmunoprecipitation and analyzed by SDS-PAGE. The c-Jun band isindicated. However, due to lower expression levels, some of the slowermigrating forms were not clearly visible.

c-Jun phosphorylation was further analyzed by two-dimensionalphosphopeptide mapping (FIG. 12C). This analysis included all theisoforms of c-Jun. All of the c-Jun specific protein bands shown in FIG.12A, isolated from equal numbers of cells, were excised from the gel andsubjected to tryptic phosphopeptide mapping. Shown is a typical result(this experiment was repeated at least three times). N-nonstimulatedcells; T-cells treated with 50 ng/ml TPA; T/A: cells: treated with 50ng/ml TPA and 1 μg/ml A23187; T/A+CsA: cells treated with T/A and 100ng/ml CsA. a,b,c,x and y correspond to the various trypticphosphopeptides of c-Jun, previously described by Boyle, et al., (Cell,64:573-584, 1991) and Smeal, et al., (Nature, 354:494-496, 1991). T1 andT2 correspond to the minor phosphorylation sites; Thr91, 93 and 95(Hibi. et al., Genes & Dev., 7:000, 1993).

While the intensity of spot b, a doubly phosphorylated tryptic peptidecontaining the C-terminal phosphorylation sites of c-Jun (Boyle, et al.,Cell, 64:573-584, 1991; Lin, et al., Cell, 70:777-789, 1992), was moreor less invariant, TPA treatment resulted in a small increase in theintensity of the monophosphorylated form of this peptide (spot c) at theexpense of the triple phosphorylated form (spot a). This effect was alsoobserved in response to costimulation with TPA+A23187. In contrast toHeLa cells and fibroblasts (Boyle, et al., supra, 1991; Minden, et al.,Nature, 1993), TPA treatment of Jurkat cells resulted in increasedphosphorylation of the N-terminal sites, corresponding to Ser63 (spot y)and Ser73 (spot x) and this effect was strongly enhanced by A23187. CsAprevented the enhancement of N-terminal phosphorylation by A23187.

EXAMPLE 12 Synergistic Activation of JNK

Studies were done to determine whether enhanced N-terminal c-Junphosphorylation in response to TPA+A23187 was due to synergisticactivation of JNK, the protein kinase that binds to c-Jun andphosphorylates its N-terminal sites. JNK exists in two forms, 46 kD and55 kD in size, both of which are activated by external stimuli (Hibi, etal., supra, 1993; Deng, et al., supra, 1993). In-gel kinase assaysindicated that both forms of JNK were activated by TPA (FIG. 13A). Wholecell extracts (WCE) of Jurkat cells incubated with TPA (T, 50 ng/ml),A23187 (A, 1 μg/ml) or CsA (100 ng/ml) for 15 minutes, alone or incombination, were separated by SDS-PAGE (100 μg protein/lane) on gelsthat were cast in the absence or presence of GST-cJun (1-223). The gelswere subjected to renaturation protocol and incubated in kinase buffercontaining γ-³²P-ATP. The protein bands corresponding to the 55 kD and46 kD forms of JNK are indicated.

While A23187 treatment by itself did not activate JNK, it potentiatedits activation by TPA. CsA blocked this costimulatory effect.

JNK can be retained on GSTcJun-glutathione (GSH) agarose affinity resinand its kinase activity measured by phosphorylation of GSTcJun. WCE (50μg) of Jurkat cells treated as described above were incubated with 5 μlof GSH agarose beads coated with 10 μg GST-cJun (1-223) for 12 hours at4° C. After extensive washing, the beads were incubated in kinase buffercontaining γ-³²P-ATP for 20 minutes at 30° C., after which the proteinswere dissociated by incubation in SDS sample buffer and separated bySDS-PAGE (FIG. 13B). The 49 kD band corresponds to GST-cJun (1-223). Thefaster migrating bands are degradation products (Hibi, et al., supra,1993).

This solid-state assay also indicated that TPA treatment resulted inactivation of JNK, which was strongly potentiated by A23187, which byitself had no effect. This synergistic activation of JNK was inhibitedby CsA (FIG. 13B). To prove that the solid-state assay measures theactivity of the same polypeptides identified by the in-gel kinase assay,JNK was first isolated on GSTcJun-GSH agarose beads and then analyzed itby an in-gel kinase assay. Both the 55 and 46 kD forms of JNK bound toGSTcJun and were regulated in the same manner revealed by the bindingassay (FIG. 13C). WCE (200 μg) of Jurkat cells treated as described inFIG. 13A were incubated with GST-cJun(1-223)-GSH agarose beads asdescribed above and the bound fraction was eluted in SDS sample bufferand separated by SDS-PAGE on a gel containing GST-cJun(1-223). The gelwas renatured and incubated in kinase buffer containing γ-³²P-ATP tolabel the JNK polypeptides.

EXAMPLE 13 Costimulation by Ca⁺⁺ is Unique to JNK and T Lymphocytes

We examined whether elevated intracellular Ca⁺⁺ affects JNK activationin other cells. JNK activity was weakly stimulated by TPA in CV1 andFR3T3 cells, but not in PC12 cells (FIG. 14). Cultures of FR3T3, CV-1,PC12 and mouse thymocytes were incubated for 15 minutes in the presenceof TPA (50 ng/ml, T), A23817 (1 μg/ml, A) and/or CsA (100 ng/ml), asindicated. WCE prepared from 2-4×10⁵ cells for the established celllines and 1.5×10⁶ cells for primary thymocytes were incubated withGSTcJun(1-223)-GSH agarose beads. After washing, JNK activity wasdetermined by solid-state phosphorylation assay as described above.

In none of these cells was JNK activity affected by A23187 or CsAtreatment. Similar results were obtained in HeLa, HepG2 and Gc cells. Bycontrast, the regulation of JNK activity in mouse thymocytes was similarto that observed in Jurkat cells. TPA induced a moderate increase in JNKactivity which was enhanced by A23187 and that costimulation wasinhibited by CsA (FIG. 14).

JNK is a proline-directed protein kinase activated by extracellularstimuli (Hibi, et al., supra, 1993). In that respect, it resembles theERK1 and 2 MAP kinases (Boulton, et al., Cell, 65:663-675, 1991). SinceERK1 and 2 appear to be involved in induction of c-fos (Gille, et al.,Nature, 358:414-417, 1992; Marais, et al., Cell, 73:381-393, 1993) andcould thereby participate in T cell activation, their regulation wasexamined. ERK1 and ERK2 activities were measured in both Jurkat andmouse thymocytes using an immunecomplex kinase assay and myelin basicprotein (MBP) as a substrate. Recombinant, kinase-defective ERK1 wasalso used a substrate for assaying MEK, the protein kinase responsiblefor activation of ERK1 and 2 (Crews, et al., Science, 258:478-480,1992). Both ERK and MEK activities were fully stimulated by TPAtreatment of either Jurkat cells or mouse thymocytes (FIG. 15).

WCE (5 μg) of Jurkat (FIG. 15, panel A) or mouse thymocytes (panel C)were incubated with 1 μg of kinase-defective ERK1 in kinase buffercontaining γ-³²P-ATP for 20 minutes. The phosphorylated proteins wereseparated by SDS-PAGE and the band corresponding to the mutant ERK1 isindicated. WCE (20 μg) of Jurkat (panel B) or mouse thymocytes (panel C)that were treated as described above were immunoprecipitated withanti-ERK antibodies (a gift from Dr. M. Weber). The immune complexeswere washed and incubated in kinase buffer containing γ-³²P-ATP and 2 μgMBP for 15 minutes at 30° C. The phosphorylated proteins were separatedby SDS-PAGE. The band corresponding to phosphorylated MBP is indicated.A23187 and CsA had no effect on either activity.

EXAMPLE 14 Synergistic Activation of JNK by Anti-CD3 and Ant-CD28

If JNK plays a central role in signal integration during T cellactivation, then other costimulatory paradigms should also cause itssynergistic activation. The regulation of JNK in response to T cellactivation with anti-CD3 and anti-CD28 antibodies was examined. Jurkatcells (1×10⁷) were incubated for 15 minutes with either normal mouseserum, 1 μg/ml anti-CD3 and/or 2 μg/ml anti-CD28, in the absence orpresence of 100 ng/ml CsA, as indicated. WCE were prepared and 100 μgsamples were analyzed for JNK activation using the in-gel kinase assay,as described above.

While incubation of Jurkat cells with either soluble anti-CD3 or solubleanti-CD28 alone had a negligible effect on JNK activity, simultaneousincubation with both antibodies resulted in strong synergisticactivation of both forms (FIG. 16A).

WCE (50 μg) of Jurkat cells treated as described above were incubatedwith GSTcJun(1-223)-GSH agarose beads and assayed for JNK activity usingthe solid-state kinase assay. The same WCE (20 μg) wereimmunoprecipitated with anti-ERK2 antibodies and assayed for MBP-kinaseactivity. CsA partially attenuated this effect. By contrast, incubationwith soluble anti-CD3 was sufficient for efficient activation of ERK2,which was not enhanced by costimulation with anti-CD28, nor was itinhibited by CsA (FIG. 16B).

To further investigate the nature of signal integration by JNK, theeffect of a suboptimal dose of TPA was examined, which by itself doesnot lead to JNK activation on the responses to either anti-CD3 oranti-CD28 (FIG. 16C). WCE (50 μg) of Jurkat cells treated as describedin Panel A with various stimuli alone or their combinations wereincubated with GSTcJun(1-223)-GSH agarose beads and assayed for JNKactivity using solid-state kinase assay. The same samples (20 μg) werealso assayed for MBP-kinase activity as described in FIG. 16B.

Together with A23187, this suboptimal dose of TPA resulted in a strongsynergistic activation of JNK but not ERK2. The activation of JNK wascompletely inhibited by CsA. The suboptimal dose of TPA also led tostrong synergistic activation of JNK together with either anti-CD3 oranti-CD28. ERK2 on the other hand, was fully activated by anti-CD3 andsuboptimal TPA, which by itself led to partial activation of ERK2 had nofurther effect. Exposure to anti-CD28 did not augment the activation ofERK2 by TPA. JNK was also efficiently activated by combined treatmentwith anti-CD3+A23187, but not by anti-CD28+A23187.

EXAMPLE 15 Activation of Ha-Ras

The effects of the various treatments on Ha-Ras activation were examinedand the results shown in FIG. 17. Jurkat cells (2×10⁶ cells per point)labeled with 0.4 mCi of ³²P-orthophosphate for 3 hours were incubatedwith nonspecific antibody (1 μg/ml mouse IgG; control), 1 μg/mlanti-CD3, 2 μg/ml anti-CD28, 10 ng/ml TPA or 500 ng/ml A23187 (A), asindicated. After 2 minutes, the cells were harvested, lysed and Ha-Raswas isolated by immunoprecipitation. The guanine nucleotide bound toHa-Ras were extracted, separated by thin layer chromatography andquantitiated as described in EXAMPLE 9. The values shown represent theaverages of two separate experiments done in duplicates. Jurkat cellswere labeled with ³²P-orthophosphate and stimulated with either TPA oranti-CD3 as described above. At the indicated time points, the cellswere harvested and the GTP content of Ha-Ras was determined as describeddirectly above.

Whereas an optimal dose of TPA and exposure to soluble anti-CD3 led toactivation of Ha-Ras, measured by an increase in its GTP content,soluble anti-CD28 had no effect on Ha-Ras activity (FIG. 17A). Theactivation of Ha-Ras by either anti-CD3 or TPA was not augmented bycostimulation with either anti-CD28 or A23187, respectively. While theactivation of Ha-Ras by TPA persisted for at least 20 minutes, theresponse to soluble anti-CD3 was highly transient (FIG. 17B). Therefore,signal integration must occur downstream of Ha-Ras.

The foregoing is meant to illustrate, but not to limit, the scope of theinvention. Indeed, those of ordinary skill in the art can readilyenvision and produce further embodiments, based on the teachings herein,without undue experimentation.

10 47 amino acids amino acid single linear peptide c-Jun/JNK bindingsite Peptide 1..47 1 Ile Leu Lys Gln Ser Met Thr Leu Asn Leu Ala Asp ProVal Gly Ser 1 5 10 15 Leu Lys Pro His Leu Arg Ala Lys Asn Ser Asp LeuLeu Thr Ser Pro 20 25 30 Asp Val Gly Leu Leu Lys Leu Ala Ser Pro Glu LeuGlu Arg Leu 35 40 45 35 base pairs nucleic acid single linear DNA(genomic) N-terminal primer CDS 1..35 2 TCTGCAGGAT CCCCATGACT GCAAAGATGGAAACG 35 34 base pairs nucleic acid single linear DNA (genomic)N-terminal primer CDS 1..34 3 TCTGCAGGAT CCCCGACGAT GCCCTCAACG CCTC 3435 base pairs nucleic acid single linear DNA (genomic) N-terminal primerCDS 1..35 4 TCTGCAGGAT CCCCGAGAGC GGACCTTATG GCTAC 35 35 base pairsnucleic acid single linear DNA (genomic) N-terminal primer CDS 1..35 5TCTGCAGGAT CCCCGCCGAC CCAGTGGGGA GCCTG 35 35 base pairs nucleic acidsingle linear DNA (genomic) N-terminal primer CDS 1..35 6 TCTGCAGGATCCCCAAGAAC TCGGACCTCC TCACC 35 30 base pairs nucleic acid single linearDNA (genomic) C-terminal primer CDS 1..30 7 TGAATTCTGC AGGCGCTCCAGCTCGGGCGA 30 33 base pairs nucleic acid single linear DNA (genomic)C-terminal primer CDS 1..33 8 TGAATTCCTG CAGGTCGGCG TGGTGGTGAT GTG 332099 base pairs nucleic acid single linear DNA (genomic) Jun CDS414..1406 9 GAATTCCGGG GCGGCCAAGA CCCGCCGCCG GCCGGCCACT GCAGGGTCCGCACTGATCCG 60 CTCCGGCGGA GAGCCGCTGC TCTGGGAAGT GAGTTCGCCT GCGGACTCCGAGGAACCGCT 120 GCGCACGAAG AGCGCTCAGT GAGTGACCGC GACTTTTCAA AGCCGGGTAGCGCGCGCGAG 180 TCGACAAGTA AGAGTGCGGG AGGCATCTTA ATTAACCCTG CGCTCCCTGGAGCGAGCTGG 240 TGAGGAGGGC GCAGCGGGGA CGACAGCCAG CGGGTGCGTG CGCTCTTAGAGAAACTTTCC 300 CTGTCAAAGG CTCCGGGGGG CGCGGGTGTC CCCCGCTTGC CAGAGCCCTGTTGCGGCCCC 360 GAAACTTGTG CGCGCACGCC AAACTAACCT CACGTGAAGT GACGGACTGTTCT ATG 416 Met 1 ACT GCA AAG ATG GAA ACG ACC TTC TAT GAC GAT GCC CTCAAC GCC TCG 464 Thr Ala Lys Met Glu Thr Thr Phe Tyr Asp Asp Ala Leu AsnAla Ser 5 10 15 TTC CTC CCG TCC GAG AGC GGA CCT TAT GGC TAC AGT AAC CCCAAG ATC 512 Phe Leu Pro Ser Glu Ser Gly Pro Tyr Gly Tyr Ser Asn Pro LysIle 20 25 30 CTG AAA CAG AGC ATG ACC CTG AAC CTG GCC GAC CCA GTG GGG AGCCTG 560 Leu Lys Gln Ser Met Thr Leu Asn Leu Ala Asp Pro Val Gly Ser Leu35 40 45 AAG CCG CAC CTC CGC GCC AAG AAC TCG GAC CTC CTC ACC TCG CCC GAC608 Lys Pro His Leu Arg Ala Lys Asn Ser Asp Leu Leu Thr Ser Pro Asp 5055 60 65 GTG GGG CTG CTC AAG CTG GCG TCG CCC GAG CTG GAG CGC CTG ATA ATC656 Val Gly Leu Leu Lys Leu Ala Ser Pro Glu Leu Glu Arg Leu Ile Ile 7075 80 CAG TCC AGC AAC GGG CAC ATC ACC ACC ACG CCG ACC CCC ACC CAG TTC704 Gln Ser Ser Asn Gly His Ile Thr Thr Thr Pro Thr Pro Thr Gln Phe 8590 95 CTG TGC CCC AAG AAC GTG ACA GAT GAG CAG GAG GGG TTC GCC GAG GGC752 Leu Cys Pro Lys Asn Val Thr Asp Glu Gln Glu Gly Phe Ala Glu Gly 100105 110 TTC GTG CGC GCC CTG GCC GAA CTG CAC AGC CAG AAC ACG CTG CCC AGC800 Phe Val Arg Ala Leu Ala Glu Leu His Ser Gln Asn Thr Leu Pro Ser 115120 125 GTC ACG TCG GCG GCG CAG CCG GTC AAC GGG GCA GGC ATG GTG GCT CCC848 Val Thr Ser Ala Ala Gln Pro Val Asn Gly Ala Gly Met Val Ala Pro 130135 140 145 GCG GTA GCC TCG GTG GCA GGG GGC AGC GGC AGC GGC GGC TTC AGCGCC 896 Ala Val Ala Ser Val Ala Gly Gly Ser Gly Ser Gly Gly Phe Ser Ala150 155 160 AGC CTG CAC AGC GAG CCG CCG GTC TAC GCA AAC CTC AGC AAC TTCAAC 944 Ser Leu His Ser Glu Pro Pro Val Tyr Ala Asn Leu Ser Asn Phe Asn165 170 175 CCA GGC GCG CTG AGC AGC GGC GGC GGG GCG CCC TCC TAC GGC GCGGCC 992 Pro Gly Ala Leu Ser Ser Gly Gly Gly Ala Pro Ser Tyr Gly Ala Ala180 185 190 GGC CTG GCC TTT CCC GCG CAA CCC CAG CAG CAG CAG CAG CCG CCGCAC 1040 Gly Leu Ala Phe Pro Ala Gln Pro Gln Gln Gln Gln Gln Pro Pro His195 200 205 CAC CTG CCC CAG CAG ATG CCC GTG CAG CAC CCG CGG CTG CAG GCCCTG 1088 His Leu Pro Gln Gln Met Pro Val Gln His Pro Arg Leu Gln Ala Leu210 215 220 225 AAG GAG GAG CCT CAG ACA GTG CCC GAG ATG CCC GGC GAG ACACCG CCC 1136 Lys Glu Glu Pro Gln Thr Val Pro Glu Met Pro Gly Glu Thr ProPro 230 235 240 CTG TCC CCC ATC GAC ATG GAG TCC CAG GAG CGG ATC AAG GCGGAG AGG 1184 Leu Ser Pro Ile Asp Met Glu Ser Gln Glu Arg Ile Lys Ala GluArg 245 250 255 AAG CGC ATG AGG AAC CGC ATC GCT GCC TCC AAG TGC CGA AAAAGG AAG 1232 Lys Arg Met Arg Asn Arg Ile Ala Ala Ser Lys Cys Arg Lys ArgLys 260 265 270 CTG GAG AGA ATC GCC CGG CTG GAG GAA AAA GTG AAA ACC TTGAAA GCT 1280 Leu Glu Arg Ile Ala Arg Leu Glu Glu Lys Val Lys Thr Leu LysAla 275 280 285 CAG AAC TCG GAG CTG GCG TCC ACG GCC AAC ATG CTC AGG GAACAG GTG 1328 Gln Asn Ser Glu Leu Ala Ser Thr Ala Asn Met Leu Arg Glu GlnVal 290 295 300 305 GCA CAG CTT AAA CAG AAA GTC ATG AAC CAC GTT AAC AGTGGG TGC CAA 1376 Ala Gln Leu Lys Gln Lys Val Met Asn His Val Asn Ser GlyCys Gln 310 315 320 CTC ATG CTA ACG CAG CAG TTG CAA ACA TTT TGAAGAGAGACCGTCGGGGG 1426 Leu Met Leu Thr Gln Gln Leu Gln Thr Phe 325 330CTGAGGGGCA ACGAAGAAAA AAAATAACAC AGAGAGACAG ACTTGAGAAC TTGACAAGTT 1486GCGACGGAGA GAAAAAAGAA GTGTCCGAGA ACTAAAGCCA AGGGTATCCA AGTTGGACTG 1546GGTTCGGTCT GACGGCGCCC CCAGTGTGCA CGAGTGGGAA GGACTTGGTC GCGCCCTCCC 1606TTGGCGTGGA GCCAGGGAGC GGCCGCCTGC GGGCTGCCCC GCTTTGCGGA CGGGCTGTCC 1666CCGCGCGAAC GGAACGTTGG ACTTTCGTTA ACATTGACCA AGAACTGCAT GGACCTAACA 1726TTCGATCTCA TTCAGTATTA AAGGGGGGAG GGGGAGGGGG TTACAAACTG CAATAGAGAC 1786TGTAGATTGC TTCTGTAGTA CTCCTTAAGA ACACAAAGCG GGGGGAGGGT TGGGGAGGGG 1846CGGCAGGAGG GAGGTTTGTG AGAGCGAGGC TGAGCCTACA GATGAACTCT TTCTGGCCTG 1906CTTTCGTTAA CTGTGTATGT ACATATATAT ATTTTTTAAT TTGATTAAAG CTGATTACTG 1966TCAATAAACA GCTTCATGCC TTTGTAAGTT ATTTCTTGTT TGTTTGTTTG GGTATCCTGC 2026CCAGTGTTGT TTGTAAATAA GAGATTTGGA GCACTCTGAG TTTACCATTT GTAATAAAGT 2086ATATAATTTT TTT 2099 331 amino acids amino acid linear protein 10 Met ThrAla Lys Met Glu Thr Thr Phe Tyr Asp Asp Ala Leu Asn Ala 1 5 10 15 SerPhe Leu Pro Ser Glu Ser Gly Pro Tyr Gly Tyr Ser Asn Pro Lys 20 25 30 IleLeu Lys Gln Ser Met Thr Leu Asn Leu Ala Asp Pro Val Gly Ser 35 40 45 LeuLys Pro His Leu Arg Ala Lys Asn Ser Asp Leu Leu Thr Ser Pro 50 55 60 AspVal Gly Leu Leu Lys Leu Ala Ser Pro Glu Leu Glu Arg Leu Ile 65 70 75 80Ile Gln Ser Ser Asn Gly His Ile Thr Thr Thr Pro Thr Pro Thr Gln 85 90 95Phe Leu Cys Pro Lys Asn Val Thr Asp Glu Gln Glu Gly Phe Ala Glu 100 105110 Gly Phe Val Arg Ala Leu Ala Glu Leu His Ser Gln Asn Thr Leu Pro 115120 125 Ser Val Thr Ser Ala Ala Gln Pro Val Asn Gly Ala Gly Met Val Ala130 135 140 Pro Ala Val Ala Ser Val Ala Gly Gly Ser Gly Ser Gly Gly PheSer 145 150 155 160 Ala Ser Leu His Ser Glu Pro Pro Val Tyr Ala Asn LeuSer Asn Phe 165 170 175 Asn Pro Gly Ala Leu Ser Ser Gly Gly Gly Ala ProSer Tyr Gly Ala 180 185 190 Ala Gly Leu Ala Phe Pro Ala Gln Pro Gln GlnGln Gln Gln Pro Pro 195 200 205 His His Leu Pro Gln Gln Met Pro Val GlnHis Pro Arg Leu Gln Ala 210 215 220 Leu Lys Glu Glu Pro Gln Thr Val ProGlu Met Pro Gly Glu Thr Pro 225 230 235 240 Pro Leu Ser Pro Ile Asp MetGlu Ser Gln Glu Arg Ile Lys Ala Glu 245 250 255 Arg Lys Arg Met Arg AsnArg Ile Ala Ala Ser Lys Cys Arg Lys Arg 260 265 270 Lys Leu Glu Arg IleAla Arg Leu Glu Glu Lys Val Lys Thr Leu Lys 275 280 285 Ala Gln Asn SerGlu Leu Ala Ser Thr Ala Asn Met Leu Arg Glu Gln 290 295 300 Val Ala GlnLeu Lys Gln Lys Val Met Asn His Val Asn Ser Gly Cys 305 310 315 320 GlnLeu Met Leu Thr Gln Gln Leu Gln Thr Phe 325 330

What is claimed is:
 1. An isolated polynucleotide encoding a c-Junpeptide consisting of about amino acid residues 33 to 79 as set forth inSEQ ID NO: 10 or conservative variations thereof.
 2. An isolatedpolynucleotide consisting of nucleotide sequence encoding amino acidresidues 33 to 79 of SEQ ID NO:10 and another nucleotide sequence.
 3. Anisolated polynucleotide encoding SEQ ID NO:
 1. 4. An isolatedpolynucleotide encoding a peptide portion of a c-Jun polypeptide havingan amino acid sequence as set forth in SEQ ID NO: 10, wherein saidpeptide portion consists of amino acid residues 33-79 of SEQ ID NO: 10and wherein said peptide portion binds a c-Jun N-terminal kinase (JNK)polypeptide.
 5. The polypeptide of claim 2, wherein the other nucleotidesequence encodes a glutathione S-transferase polypeptide.
 6. Thepolynucleotide of claim 1, wherein the polynucleotide is in a vector. 7.The polynucleotide of claim 6, wherein the vector is a plasmid.
 8. Thepolynucleotide of claim 6, wherein the vector is a virus-derived vector.9. A host cell containing a vector of claim
 6. 10. A method forproducing a peptide of SEQ ID NO:1 comprising: a) culturing a host cellof claim 9 under conditions which allow expression of thepolynucleotide; and b) obtaining the peptide of SEQ ID NO:1.